CN113350278B

CN113350278B - Stable aqueous antibody formulations

Info

Publication number: CN113350278B
Application number: CN202110799711.XA
Authority: CN
Inventors: W·利奇; R·勒乌斯; J·麦克吉夫尼; K·纽厄尔; K·D·斯图尔特
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2013-10-24
Filing date: 2014-10-23
Publication date: 2023-03-24
Anticipated expiration: 2034-10-23
Also published as: HUE055919T2; KR20160068946A; US20150118249A1; AU2014339984A1; CN112107538A; HK1221900A1; EP3060229A1; US20200297855A1; CN113350278A; AU2022291595A1; SG11201603206UA; RU2016119755A; US20190201535A1; CA2926089A1; PL3060229T3; EP3892288A1; LT3060229T; MX2016004605A; BR112016008576B1; RU2021137159A

Abstract

The present invention relates to stable aqueous antibody formulations. In some embodiments, the stable aqueous formulation comprises about 2mg/mL to about 100mg/mL of the anti-IL 5R antibody, and about 0.002% to about 0.01% polysorbate-20. The invention also provides methods of making and methods of using such antibody formulations.

Description

Stable aqueous antibody formulations

The present application is a divisional application of chinese patent application 201480055263.0 "stable aqueous antibody formulation" filed as 2014, 10, 23.

Technical Field

The present invention relates to stable aqueous antibody formulations. In some embodiments, the stable aqueous formulation comprises: about 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by Kabat (Kabat) of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by Kabat of SEQ ID NOs 8-10; and about 0.002% to about 0.01% polysorbate-20. The invention also provides methods of making and methods of using such antibody formulations.

Background

Antibodies have been used to treat various diseases and disorders due to the specificity of their target recognition, thereby producing highly selective results after systemic administration. In order to keep an antibody effective, the antibody must maintain its biological activity during production, purification, transport and storage. New production and purification techniques have been developed to allow the production of large quantities of highly purified monoclonal antibodies. However, there remains the problem of stabilizing these antibodies for transport and storage, and even the problem of providing the antibodies in a dosage form suitable for administration.

Denaturation, aggregation, contamination and particle formation can constitute significant obstacles in antibody formulation and storage. Due to the wide variety of antibodies, there is no universal formulation or condition suitable for storage of all antibodies. The optimal formulation and conditions for the storage of an antibody are generally specific to the antibody. Thus, antibody storage formulations and methods are often an important part of the commercial antibody research and development process.

Different approaches have been proposed to overcome the difficulties associated with antibody stability. For example, in some cases, antibodies are often lyophilized and then reconstituted shortly before administration. However, rehydration is generally undesirable as it adds an additional step in the administration process and may introduce contaminants to the formulation. In addition, even rehydrated antibodies may suffer from aggregation and particle formation. Accordingly, there is a need to provide stable aqueous antibody formulations that can overcome the difficulties associated with shipping and storage.

Disclosure of Invention

The present invention relates to a stable aqueous antibody formulation comprising: (a) About 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the aqueous formulation further comprises an uncharged excipient. In some embodiments, the uncharged excipient is trehalose. In some embodiments, the concentration of the uncharged excipient is about 20mM to about 80mM. In some embodiments, the concentration of the uncharged excipient is about 200mM to about 400mM.

The antibody may be present in various concentrations. In some embodiments, the formulation comprises about 2 to about 20mg/ml of the antibody. In some embodiments, the formulation comprises about 20 to about 100mg/ml of the antibody. In one embodiment, the formulation comprises 30mg/ml of the antibody.

The formulation may further comprise arginine. In some embodiments, the arginine is L-arginine. In some embodiments, the formulation comprises about 100mM to about 200mM L-arginine. In some embodiments, the formulation comprises about 120mM to about 140mM L-arginine, and about 40mM to about 60mM uncharged excipient. In one embodiment, the formulation comprises about 125mM L-arginine. In one embodiment, the formulation comprises about 130mM L-arginine.

In some embodiments, the formulation further comprises histidine. In some embodiments, the concentration of histidine is about 15mM to about 30mM. In one embodiment, the concentration of histidine is about 20mM.

In some embodiments, the antibody is not subjected to lyophilization.

In some embodiments, the invention relates to a stable aqueous antibody formulation comprising about 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein said formulation is stable upon storage at about 40 ℃ for at least 1 month. In some embodiments, the formulation is stable upon storage at about 25 ℃ for at least 3 months. In some embodiments, the formulation is stable upon storage at about 5 ℃ for at least 18 months. In some embodiments, an antibody stored at about 40 ℃ for at least 1 month retains at least 80% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 5 ℃ for at least 6 months retains at least 80% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 40 ℃ for at least 1 month retains at least 95% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 5 ℃ for at least 6 months retains at least 95% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored. In some embodiments, less than 2% of the antibody forms an aggregate upon storage at about 40 ℃ for at least 1 month, as determined by HPSEC. In some embodiments, less than 2% of the antibody forms an aggregate after storage at about 5 ° for at least 12 months, as determined by HPSEC.

In some embodiments, the formulation is substantially free of particles after storage at about 40 ℃ for at least 1 month, as determined by visual inspection. In some embodiments, the formulation is substantially free of particles after storage at about 5 ℃ for at least 12 months, as determined by visual inspection.

In some embodiments, the formulation is an injectable formulation. In some embodiments, the formulation is suitable for intravenous, subcutaneous, or intramuscular administration.

In some embodiments, the invention relates to a sealed container containing an antibody formulation as described herein. In some embodiments, the invention relates to a pharmaceutical unit dosage form suitable for parenteral administration to a human comprising an antibody formulation as described herein in a suitable container. In some embodiments, the antibody formulation is administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the suitable container is a pre-filled syringe. In some embodiments, the pre-filled syringe comprises an injection needle. In some embodiments, the injection needle is a 29G thin tube injection needle. In some embodiments, the pre-filled syringe is a plastic syringe or a glass syringe. In some embodiments, the pre-filled syringe is made of a material that is substantially free of tungsten.

In some embodiments, the pre-filled syringe is coated with silicone. In some embodiments, the prefilled syringe comprises a plunger having a disk of fluoropolymer resin. In some embodiments, the pre-filled syringe comprises: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) about 0.002% to about 0.01% polysorbate-20. In some embodiments, the pre-filled syringe further comprises: (c) About 40mM to about 60mM trehalose, and (d) about 110mM to about 150mM L-arginine. In some embodiments, the present invention relates to a pre-filled syringe comprising: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) about 0.002% to about 0.01% polysorbate-20. In some embodiments, the formulation further comprises: (c) about 200mM to about 300mM trehalose.

In some embodiments, the invention relates to a kit comprising a formulation described herein, a container described herein, a unit dosage form described herein, or a pre-filled syringe described herein.

In some embodiments, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.002% to about 0.01% polysorbate-20; (c) about 40mM to about 60mM trehalose; (d) about 110mM to about 150mM L-arginine; and about 15mM to about 30mM histidine. In one embodiment, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 50mM trehalose; (d) about 130mM L-arginine; and (e) about 20mM histidine.

In some embodiments, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.002% to about 0.01% polysorbate-20; (c) about 200mM to about 300mM trehalose; and (d) about 15mM to about 30mM histidine. In one embodiment, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 250mM trehalose; and (d) about 20mM histidine. In another embodiment, the invention relates to a stable aqueous antibody formulation comprising: (a) About 30mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 250mM trehalose; and (d) about 20mM histidine.

In some embodiments, the present invention relates to a method of preparing a stable aqueous antibody formulation comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) Placing the isolated antibody in a stabilizing formulation to form the stable aqueous antibody formulation, wherein the resulting stable aqueous antibody formulation comprises: (i) About 2mg/mL to about 100mg/mL of an antibody and (ii) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the present invention relates to a method of preparing a stable aqueous antibody formulation comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) Diluting the antibody to about 2mg/mL to about 20mg/mL of antibody in a solution comprising: (i) about 0.002% to about 0.01% polysorbate-20; (ii) about 40mM to about 60mM trehalose; and (iii) about 110mM to about 150mM L-arginine.

In some embodiments, the present invention relates to a method of preparing a stable aqueous antibody formulation comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) Diluting the antibody to about 20mg/mL to about 100mg/mL of antibody in a solution comprising: (i) about 0.002% to about 0.01% polysorbate-20; and (ii) about 200mM to about 300mM trehalose.

In some embodiments, the present invention relates to a method of making a reconstituted antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, the method comprising: (a) purifying the antibody from the cell culture; (b) lyophilizing the isolated antibody; (c) Adding the lyophilized antibody to an aqueous solution to form a reconstituted antibody formulation, wherein the reconstituted antibody formulation comprises: (i) About 2mg/mL to about 100mg/mL of an antibody and (ii) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of particles. In some embodiments, the antibody formulation comprises an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of active glutathione S-transferase (GST). In some embodiments, the antibody formulation is substantially free of GST. In some embodiments, the antibody formulation is substantially free of particles when stored at 38 ℃ -42 ℃ for at least 1 month. In some embodiments, the antibody formulation is substantially free of particles when stored at 2 ℃ -6 ℃ for at least 6 months. In some embodiments, the antibody formulation is substantially free of particles when stored at 2 ℃ -6 ℃ for at least 18 months.

In some embodiments, the invention relates to a method of purifying an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, the method comprising: (a) obtaining a cell culture comprising the antibody; (b) performing affinity chromatography on the antibody; (c) performing cation exchange on the antibody; (d) performing mixed mode chromatography on the antibody. In some embodiments, the method further comprises a virus inactivation process and/or a diafiltration process.

In some embodiments, the invention relates to a method of treating a pulmonary disease or disorder in a subject, the method comprising administering a therapeutically effective amount of an antibody formulation described herein, a container described herein, a unit dosage form described herein, or a pre-filled syringe described herein. In some embodiments, the pulmonary disease or disorder is an eosinophilic disease or disorder. In some embodiments, the pulmonary disease or disorder is asthma, COPD, eosinophilic asthma, eosinophilic and neutrophilic asthma, aspirin-sensitive asthma, allergic bronchopulmonary aspergillosis, acute and chronic eosinophilic bronchitis, acute and chronic eosinophilic pneumonia, churg-schii's syndrome, hypereosinophilic syndrome, drug, irritant and radiation-induced hypereosinophilia, infection-induced hypereosinophilia (fungi, tuberculosis, parasites), autoimmune-related hypereosinophilia, eosinophilic esophagitis, crohn's disease, or a combination thereof. In some embodiments, the pulmonary disease or disorder is asthma. In some embodiments, the pulmonary disease or disorder is Chronic Obstructive Pulmonary Disease (COPD).

Drawings

FIG. 1 amino acid sequence of anti-IL 5R antibody.

FIG. 2 shows the effect of polysorbate-20 in solution on the monomer fraction. A polysorbate concentration higher than 0.005% is required to fully maintain the monomer level.

FIG. 3 shows the effect of polysorbate-20 in 2g/L solution on sub-visible particle counts. Data for particles >2 μm are not shown, but show a similar pattern as larger particles. The data indicate that the sub-visible particle level in the 2g/L solution was not controlled by any polysorbate level.

FIG. 4 shows the effect of polysorbate-20 on sub-visible particle counts in a 100g/L solution. Data for particles >2 μm are not shown, but show a similar pattern as larger particles. The data indicate that levels of polysorbate above 0.003% in 100g/L solution control sub-visibility particle levels.

FIG. 5 HPLC monomers (%) and others (%) as a function of solution pH and protein concentration. Monomer loss is minimized in the pH range of 5.5-6.5.

Figure 6 particle formation as a function of solution pH and protein concentration, including sub-visible particles >10 μm as measured by MFI and visible particles evaluated by comparison to standards. Sub-visible particle counts were dependent on protein concentration, but showed no trend with pH. More visible particles were observed in the lower protein concentration solutions in the pH range of 5.5-6.5.

Figure 7 sub-visibility particle counts >10 μm filtered by aspect ratio to remove silicone oil droplets. This data compares the SVP counts immediately after shipping to those after 1 month of storage at 25 ℃. Independent of PS-20, high counts were observed for low protein concentrations.

Figure 8 particle counts >10 μm after simulated transport determined by MFI for different protein concentrations and formulations. The higher ionic strength formulations are more stable, as are >10 g/L trehalose formulations.

Figure 9 particle counts >10 μm after simulated transport by MFI assay for 2g/L protein and different formulations. Arginine concentration should be >50mM and NaCl concentration should be >75mM.

Figure 10 particle counts >10 μm after simulated transport by MFI assay for 2g/L protein and different formulations. Any excipient concentration within this range is acceptable.

Figure 11 particle count after simulated transport by MFI assay >10 μm for 2g/L protein and different formulations.

FIG. 12 shows monomer loss for 2mg/mL, 20mg/mL, and 100mg/mL formulations in vials and pre-filled syringes. All PFSs showed similar losses to vials and to each other.

FIG. 13 is a graphical representation showing interval strategy. Blue indicates arginine or NaCl containing formulations. Green indicates trehalose formulation. The data points are prepared samples and the lines indicate the interval options available for obtaining intermediate doses.

FIG. 14. Test plan for stability study # 2. All tests labeled were performed on antibody formulations. Yellow shading indicates the tests to be performed on placebo. The symbol "ABC" indicates the submissions of the following tests: potency (bioassay), RP-HPLC, cIEF, non-reduced bioanalyzer, and reduced bioanalyzer.

Figure 15 is a graph showing samples prepared to define a design space as a function of protein concentration and polysorbate-20 ("PS-20") concentration for two formulation intervals.

Fig. 16 is a graph of sub-visibility particles at time 0 as measured by MFI after delivery. The results show that particle formation was observed if PS-20 was not present, but 0.002% PS-20 was sufficient to inhibit particle formation after shipping.

Fig. 17 (a-D) visible particle observations were scored against an appearance criterion and shown here at the 9 th month time point. The observation was performed in close proximity to the light. The samples shown included a pre-filled syringe ("PFS") containing a trehalose formulation (fig. 17A), a vial containing a trehalose formulation (fig. 17B), a PFS containing an arginine formulation (fig. 17C), and a vial containing an arginine formulation (fig. 17D). This data supports the goal of 0.006% -PS-20 and the acceptable range of 0.002% -0.01% -PS-20 in PFS. Vials are shown as worst case comparisons.

FIG. 18 compares the different SVP methods used to capture the sample time increase with the visible particle association. Flow cytometry and small particle counts (> 1 μm and >2 μm) determined by MFI can also capture this trend.

Figure 19 is a comparison of appearance criterion scores and MFI results (> 1 μm particles) for trehalose formulations in PFS (figure 19A) and vials (figure 19B). Good agreement was observed for both methods, which both indicate that the acceptable PS-20 range is 0.002% -0.01%.

Figure 20 represents the interval design as outlined in example 3 and the run-ahead batch stability study performed in ABC. The orange shaded area indicates arginine formulation and the blue shaded area indicates trehalose formulation, all having 0.006% ps-20.

FIG. 21 shows an illustrative example of the antibody decontamination process.

FIG. 22 shows a flow-through 2D gel analysis of a protein A column used in anti-IL 5R antibody purification.

Detailed description of the preferred embodiments

It should be appreciated that the specific implementations shown and described herein are examples and are not intended to otherwise limit the scope of the present application in any way. It should also be appreciated that the various embodiments and features of the invention described herein may be combined in any and all ways.

The patents, patent applications, web sites, company names, and scientific literature referred to herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Also, any conflict between a definition in the art of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

As used in this specification, the singular forms "a", "an" and "the" include specifically also the plural forms of the terms in which they are referred to, unless the content clearly dictates otherwise.

Throughout this disclosure, all percentages, ratios, and the like are expressed as "by weight" unless otherwise indicated. As used herein, "by weight" is synonymous with the term "by mass" and indicates that the ratios or percentages defined herein are expressed in terms of weight, rather than volume, thickness, or some other measure.

The term "about" is used herein to mean about (approximate), in the vicinity of (8230) \8230; (in the region of), roughly (roughly), or left or right (around). When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values set forth. Generally, the term "about" is used herein to modify a numerical value by a variance of 10% above and below the stated value.

Technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application relates, unless otherwise defined. Reference is made herein to various methods and materials known to those of ordinary skill in the art. Standard references which set forth the general principles of recombinant DNA technology include sambrook et al, "molecular cloning: a Laboratory Manual ", 2nd edition, cold Spring Harbor Laboratory, new York (1989) (Sambrook et al," Molecular Cloning: A Laboratory Manual, "2nd Ed., cold Spring Harbor Laboratory Press, new York (1989)); codaft et al, handbook of molecular and Cellular Methods in Medicine, CRC Press, bocardon (1995) (Kaufman et al, eds., "Handbook of molecular and Cellular Methods in Biology in Medicine," CRC Press, boca Raton (1995)); and macpherson, directed mutagenesis: utility methods ", IRL Press, oxford (1991) (McPherson, ed.," Directed Mutagenesis: A Practical Approach, "IRL Press, oxford (1991)), the disclosure of each of these works is incorporated herein by reference in its entirety.

The present invention relates to stable aqueous antibody formulations. As described herein, the term "antibody formulation" refers to a composition comprising one or more antibody molecules. The term "antibody" in the present invention is not particularly limited. For the sake of clarity, "antibody" is taken in its broadest sense and includes any immunoglobulin (Ig), an active or desired variant thereof, and an active or desired fragment thereof (e.g., fab fragments, camelid antibodies (single chain antibodies), and nanobodies). The term "antibody" may also refer to dimers or multimers. Antibodies may be polyclonal or monoclonal, and may be naturally occurring or recombinantly produced. Thus, human, non-human, humanized and chimeric antibodies are included within the term "antibody". Typically, the antibody is a monoclonal antibody of one of the following classes: igG, igE, igM, igD, and IgA; and more typically IgG or IgA.

The antibodies of the invention may be from any animal source, including birds and mammals. In some embodiments, the antibodies of the methods of the invention are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins. See, for example, U.S. Pat. No.5,939,598 to Kucherlapati (Kucherlapati) et al.

Antibodies of the invention can include, e.g., natural antibodies, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments (e.g., antibody fragments that bind and/or recognize one or more antigens), humanized antibodies, human antibodies (Jacobowetz et al, proc. Natl.Acad.Sci.USA 90 (1993)); jacobowetz et al, natl.Acad.Sci.USA 90, 255-258 (1993)), bulugmann et al, annu.7 (1993) (Bruggemann et al, yeast in munol.7:33 (1993)), U.S. Pat. No.5,591,669 and 5,545,807), antibodies and antibody fragments isolated from phage libraries (Mackoff et al, nature 552-554 (1990) (Mcfffery et al, nature 1997810, 199779, 1985,628, 1985,775,628, 1985,779, markset al, markutson et al, markokutakutaki et al, 19831 (199779, 1995-775, 199775, 1990)) (Biokutson). Antibodies purified by the methods of the invention can be recombinantly fused to the N-or C-terminus of a heterologous polypeptide or chemically conjugated (including covalently and non-covalently conjugated) to a polypeptide or other composition. For example, antibodies purified by the methods of the invention can be recombinantly fused or conjugated to molecules that can be used as tags in detection assays and effector molecules, such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publication WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. nos. 5,314,995; and EP 396,387.

In some embodiments, the antibody formulations of the invention comprise an anti-IL 5 receptor (anti-IL 5R) antibody. The antibodies of the invention specifically bind to the antibody or fragment thereof of interest and do not specifically bind to other antigens or fragments thereof. For example, an anti-IL 5R antibody will immunospecifically bind to an interleukin-5 receptor polypeptide and not specifically bind to other polypeptides. Preferably, the antibody or antibody fragment that immunospecifically binds to the IL-5 receptor has a higher affinity for the IL-5 receptor or a fragment of the IL-5 receptor polypeptide than for the other polypeptide or other polypeptide fragment. The affinity of an antibody is a measure of its binding to a specific antigen at a single antigen-antibody site, and is essentially the sum of all attractive and repulsive forces present in the interaction between the antibody and the antigen-binding site of a particular epitope. The affinity of an antibody for a particular antigen (e.g., an IL-5 polypeptide or a fragment of an IL-5 polypeptide) can be represented by the equilibrium constant K, which is represented by the equation K = [ Ag Ab = [)]/[Ag][Ab]By definition, is the affinity of the binding site of an antibody, wherein [ Ag]Is the concentration of free antigen, [ Ab ]]Is the concentration of free antibody and [ Ag Ab]Is the concentration of the antigen-antibody complex. When the antigen and antibody react strongly together there will be very little free antigen or free antibody, and thus anti-The equilibrium constant or affinity of the body will be high. High affinity antibodies arise when there is good matching between antigen and antibody (for a discussion of antibody affinity, see Segal and Ron eds, 1994, immunological and Inflammation-Basic Mechanisms and Clinical outcomes, mcGraw-Hill, inc., new York, pp 56-57 (Sigal and Ron ed.,1994, immunology and Inflammation-Basic Mechanisms and Clinical sequences, mcGraw-Hill, inc. New York protocols 56-57), and Western Mo et al,1995, introduction to immunology-Health Sciences, mcGray-Hill Book, australia, pp 31-32 (Seymour et ah,1995, immunology-Introduction for the Health Sciences, graw-Hik Company, booStrand tissues 31-32)). Preferably, the antibody or antibody fragment that immunospecifically binds to an IL-5 polypeptide or fragment thereof does not cross-react with other antigens. That is, the antibody or antibody fragment immunospecifically binds to an IL-5 polypeptide or fragment thereof with a higher energy than to other polypeptides or fragments of other polypeptides (for a discussion of antibody specificity, see, e.g., paul eds., 1989, basic immunology, 2nd edition, utility Press, new York, pp. 332-336 (Paul ed.,1989, functional immunology, 2) ^nd ed., raven Press, new York atpages 332-336)). Antibodies or antibody fragments that immunospecifically bind to An IL-5 polypeptide can be identified, for example, by immunoassays such as Radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), and BIAcore assays or other techniques known to those skilled in the art (see, e.g., western Moore et al,1995, introduction to immunology-Health Sciences, mgelo-Hill Book, australia, pp.33-41 (Seymour et al,1995, immunology-An Introduction for the Health Sciences, mcGraw-Hill Book Company, australia at pages 33-41)). An antibody or antibody fragment that immunospecifically binds to an IL-5 polypeptide or fragment thereof is only counterproductive to the IL-5 polypeptide and does not significantly counterproduct to other activities.

In one embodiment, the IL-5R polypeptide is human IL-5R, an analog, derivative, or fragment thereof. The nucleotide sequence of human IL-5R can be found in the GenBank database (see, e.g., accession number M96652.1). The amino acid sequence of human IL-5R can be found in the GenBank database (see, e.g., accession number Q01344). Each of these accession numbers is expressly incorporated herein by reference.

In some embodiments, the antibody formulation comprises an anti-IL 5R antibody, e.g., a human anti-IL 5R antibody. In some embodiments, the anti-IL 5R antibody comprises: a light chain comprising SEQ ID NO 2 and a heavy chain comprising SEQ ID NO 4. In other embodiments, the anti-IL 5R antibody comprises: the light chain variable region comprising SEQ ID NO 1 and the heavy chain variable region comprising SEQ ID NO 3. In another embodiment, the anti-IL 5R antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 8-10. One of ordinary skill in the art will be readily able to identify the georgia (Chothia) defined, ebeam (Abm) defined, or other CDRs.

In one embodiment, the anti-IL 5R antibody is benralizumab. Information regarding benralizumab (or a fragment thereof) for use in the methods provided herein can be found in U.S. patent application publication No. US 2010/0291073 A1, the disclosure of which is incorporated herein by reference in its entirety.

As used herein, the term "analog" or "antibody analog" in the context of an antibody refers to a second antibody, i.e., an antibody analog, that has a function similar to or identical to an antibody, but does not necessarily comprise an amino acid sequence similar to or identical to an antibody or possess a structure similar to or identical to an antibody. An antibody having a similar amino acid sequence refers to an antibody analog that satisfies at least one of: (a) An antibody analog having an amino acid sequence at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody; (b) An antibody analog encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding an antibody having at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, or at least 150 consecutive amino acid residues; and (c) an antibody analog encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a nucleotide sequence encoding the antibody. An antibody analog having a structure similar to an antibody refers to a proteinaceous agent having a secondary, tertiary, or quaternary structure similar to an antibody. Antibody analogs or antibody structures can be determined by methods known to those skilled in the art, including but not limited to peptide sequencing, X-ray diffraction crystallography, nuclear magnetic resonance, circular dichroism, and crystal electron microscopy.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are said to be identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity = number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm for comparing two sequences is Karlin and alchol, 1990, proceedings of the american academy of sciences 87-2264-2268 (Karlin and Altschul,1990, proc.natl.acad.sci.u.s.a.87. This algorithm is incorporated into the NBLAST and XBLAST programs of alchol et al, 1990, journal of molecular biology 215 (Altschul et ah,1990, j.mol. Biol.215, 403). BLAST nucleotide searches are performed using NBLAST nucleotide program parameter settings such as score =100, word length =12, in order to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed using XBLAST program parameter settings, such as score =50, word length =3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gap alignments for comparison purposes, the gap BLAST programs described in Aluchul et al,1997, nucleic Acids research 25, 3389-3402 (Altschul et al,1997, nucleic Acids Res.25. Alternatively, an iterative search can be performed using PSI-BLAST to detect distant relationships between molecules (supra). When utilizing the BLAST program, the gapped BLAST program, and the PSI-BLAST program, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Meiers and Miller,1988, CABIOS 4. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In some embodiments, the antibodies in the antibody formulation are purified prior to addition to the antibody formulation. The terms "isolate" and "purify" refer to separating an antibody from impurities or other contaminants in a composition in which the antibody is present (e.g., a composition comprising host cell proteins). In some embodiments, at least 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% (w/w) of the impurity is purified away from the antibody. For example, in some embodiments, purification of an antibody, e.g., an anti-IL 5R antibody, comprises separating the antibody from 99% (w/w) of the host cell proteins originally present in the composition.

In some embodiments, the terms "isolate" and "purify" refer to separating an antibody (e.g., an anti-IL 5R antibody) from impurities or other contaminants in a composition to an extent consistent with the guidelines of a governmental organization (e.g., the world health organization or the U.S. food and drug administration).

Methods for purifying antibodies are known to those skilled in the art. Suitable techniques for performing purification include different types of chromatography, such as affinity chromatography, hydrophobic interactions, ion exchange (such as cation exchange chromatography or mixed mode chromatography), and diafiltration.

Affinity chromatography refers to a separation method of an affinity ligand that binds an antibody to the antibody by virtue of its specific binding properties. Functional affinity ligands may be immobilized on a solid or semi-solid support such that when a composition comprising the antibody passes over the ligand and solid support, the antibody having specific binding affinity for the ligand is adsorbed onto the ligand and one or more other impurities are not adsorbed (or bind with lower affinity) and can be separated from the antibody. Examples of impurities that do not typically bind (or bind poorly) include process-related impurities (e.g., host cell proteins, DNA, media components) and some product-related impurities (e.g., antibody fragments). In some embodiments, the solid support comprising the ligand is washed one or more times with a buffer to remove additional impurities, after which the adsorbed antibody is removed from the ligand and the support. After one or more impurities have been removed, the adsorbed antibody can be removed (eluted) from the ligand and support, resulting in separation of the antibody from the original composition. Methods of removing antibodies from the ligand and support depend on the ligand and are known to those skilled in the art and may include, for example, changes in environmental aspects (e.g., pH), addition of chaotropic or denaturing agents, or addition of commercially available elution buffers. In some embodiments, more than one affinity purification process can be used on a composition. Different affinity ligands are known in the art, including protein a and protein G (and combinations thereof). Immobilized ligands are commercially available. For example, protein A affinity systems include MabSelect, mabSelect Sure, mabSelect Xtra, mabSelect Sure LX, sepharose CL-4B, proSep vA Ultra, and Ceramic HyperD.

Ion exchange chromatography includes cation exchange chromatography and mixed chromatography. Cation exchange chromatography refers to any method by which an antibody and some impurity or impurities can be separated based on charge differences using a cation exchange matrix. The cation exchange matrix generally comprises covalently bound negatively charged groups. Weak or strong cation exchange resins may be used. Typically, strong cation exchange resins include supported organic groups that contain sulfonic acid or sulfonate groups (depending on pH). Weak cation exchange resins typically include supported organic groups that contain carboxylic acid or carboxylate groups (depending on pH). In certain embodiments, multimodal cation exchange resins may be used that incorporate additional binding mechanisms as well as ionic interactions, such as one or more of hydrogen bonding interactions and hydrophobic interactions. Examples of suitable cation exchange resins are well known in the art and may include, but are not limited to, fractogel, carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S), PROPAC WCX-10 ^TM (Dionex), capto S, S-Cross-Linked agarose FF, fractogel EMD SO ₃ M, toyopearl Megacap II SP 550C, poros 50HS, and SP-Cross-Linked agarose matrices. In some embodiments, more than one cation exchange chromatography process may be used on the composition.

Mixed mode chromatography refers to methods that utilize more than one form of interaction between a stationary phase and an analyte in order to achieve their separation from impurities (e.g., process-related impurities such as host cell proteins, DNA, and/or endogenous or exogenous viruses). Examples of suitable anion exchange matrices are known in the art and may include, but are not limited to, capto Adhere, sartobind Q, natrix Q, chromasorb Q, and Mustang Q.

In some embodiments, additional filtration steps may be used to remove impurities. For example, in some embodiments, nanofiltration or ultrafiltration is used. Nanofiltration involves passing the composition through a matrix having a pore size of, for example, less than 75nm, less than 50nm, and even less than 15nm, to separate impurities, such as viruses, from the antibodies. Commercially available nanofilters and ultrafilters that can be used are produced by different suppliers, such as Millipore (Billerica, massachusetts, e.g., viresolve Pro and Viresolve Pro +), pall Corporation (East mountain, N.Y.) (Pall Corporation (East Hills, N.Y.)), GE medical science (Piscatavir, N.J.)), and Saedori Corporation (Gottingen, germany) (Sartorius Corporation (Goettingen, germany)).

In some embodiments, an antibody of the invention (e.g., an anti-IL 5R comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 8-10) is 1mg/ml to 200mg/ml, 2mg/ml to 100mg/ml, 2mg/ml to 30mg/ml, 2mg/ml to 25mg/ml, 2mg/ml to 20mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml or 20mg/ml. In some embodiments, an antibody of the invention, e.g., anti-IL 5R, is at a concentration of about 20mg/ml, 25mg/ml, 30mg/ml, 40mg/ml, 45mg/ml, 50mg/ml, 55mg/ml, 60mg/ml, 65mg/ml, 70mg/ml, 75mg/ml, 80mg/ml, 85mg/ml, 90mg/ml, 95mg/ml, or 100mg/ml.

The antibody formulations of the invention may comprise uncharged excipients. The term excipient refers to a pharmacologically inactive substance formulated with the antibodies described herein. In some embodiments, the excipient may help prevent denaturation or otherwise help stabilize the antibody. Suitable excipients that can be used in pharmaceutical compositions are well known in the art. Examples may be taken from e.g. handbooks: pyrnalo, arsolo R: "Pharmaceutical science of Remington", michkoppen, iston, pa.,1990 (Gennaro, alfonso R.: remington's Pharmaceutical Sciences ", mack Publishing Company, easton, pa., 1990). In some embodiments, the excipient is an "uncharged" excipient, i.e., the excipient does not carry a positive "+" charge or a negative "-" charge. In some embodiments, the excipient is selected from the group consisting of: fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, dextran, amylase, dextrin, cyclodextrin, soluble starch, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, hydroxyethyl starch, water-soluble dextran.

In some embodiments, in the antibody formulation, the uncharged excipient is about 1mM to about 1M, about 2mM to about 500mM, about 5mM to about 400mM, about 10mM to about 300mM, or about 20mM to about 250mM. In some embodiments, in an antibody formulation (e.g., an antibody formulation comprising 2 to 20mg/mL antibody), the uncharged excipient is about 5mM to about 150mM, about 10mM to about 100mM, about 20mM to about 80mM, about 30mM, about 40mM, about 50mM, about 60mM, or about 70mM. In one embodiment, in the antibody formulation, the uncharged excipient is about 50mM. In some embodiments, in an antibody formulation (e.g., an antibody formulation comprising 20 to 100mg/mL antibody), the uncharged excipient is about 50mM to about 800mM, about 100mM to about 500mM, about 150mM to about 400mM, about 200mM, about 300mM, or about 250mM. In one embodiment, in the antibody formulation, the uncharged excipient is about 250mM.

In some embodiments, the uncharged excipient is trehalose, as represented by the formula:

in some embodiments, in the antibody formulation, the trehalose is about 1mM to about 1M, about 2mM to about 500mM, about 5mM to about 400mM, about 10mM to about 300mM, or about 20mM to about 250mM. In some embodiments, in an antibody formulation (e.g., an antibody formulation comprising 2 to 20mg/mL antibody), the trehalose is about 5mM to about 150mM, about 10mM to about 100mM, about 20mM to about 80mM, about 30mM, about 40mM, about 50mM, about 60mM, or about 70mM. In one embodiment, in the antibody formulation, the trehalose is about 50mM. In some embodiments, in an antibody formulation (e.g., an antibody formulation comprising 20 to 100mg/mL antibody), the trehalose is about 50mM to about 800mM, about 100mM to about 500mM, about 150mM to about 400mM, about 200mM, about 300mM, or about 250mM. In one embodiment, in the antibody formulation, the trehalose is about 250mM.

The antibody formulations of the present invention comprise arginine. Arginine is a conditional nonessential amino acid that can be represented by the following formula:

as used herein, arginine may include arginine in free base form, as well as all salts thereof. In some embodiments, arginine includes pharmaceutically acceptable salts thereof. For example, arginine will include arginine hydrochloride. As used herein, arginine also includes all enantiomers (L-arginine and S-arginine), as well as any combination of enantiomers (e.g., 50% L-arginine and 50% S-arginine; 90% -100% L-arginine and 10% -0% S-arginine, etc.). In some embodiments, the term "arginine" includes greater than 99% L-arginine and less than 1% S-arginine. In some embodiments, the term "arginine" includes enantiomerically pure L-arginine. In some embodiments, the arginine is pharmaceutical grade arginine.

Arginine may be present at various concentrations in the antibody formulation. In some embodiments, the antibody formulation comprises greater than 50mM arginine, greater than 75mM arginine, greater than 100mM arginine, greater than 125mM arginine, greater than 130mM arginine, greater than 150mM arginine, greater than 175mM arginine, or greater than 200mM arginine.

In some embodiments, the antibody formulation comprises up to 800mM arginine, up to 600mM arginine, up to 400mM arginine, up to 200mM arginine, up to 150mM arginine, up to 130mM arginine, or up to 125mM arginine. In some embodiments, the antibody formulation comprises 50mM to 300mM, 75mM to 250mM, 100mM to 200mM, 110mM to 160mM, 120mM to 150mM, or about 125mM arginine. In some embodiments, the antibody formulation comprises 125mM arginine. In some embodiments, the antibody formulation comprises 130mM arginine. In some embodiments, arginine is added in an amount sufficient to maintain the osmolality of the formulation. In some embodiments, arginine is added in an amount sufficient to obtain a hypertonic solution. Applicants have found that in some embodiments, the increased ionic strength of the antibody formulation provides increased stability and reduction in particle formation.

The antibody formulations described herein may have different viscosities. Methods of measuring viscosity of antibody formulations are known to those skilled in the art and may include, for example, a rheometer (e.g., an Anton Paar MCR301 rheometer with 50mm, 40mm, or 20mm plate attachments). In some embodiments of the invention, the viscosity is reported at a high shear limit of a shear rate of 1000 per second. In some embodiments, the antibody formulation has a viscosity of less than 20 centipoise (cP), less than 18cP, less than 15cP, less than 13cP, or less than 11 cP. In some embodiments, the antibody formulation has a viscosity of less than 13 cP. One skilled in the art will appreciate that viscosity is temperature dependent, and thus unless otherwise stated, the viscosities provided herein are measured at 25 ℃ unless otherwise stated.

The term "injection force" is the amount of pressure (newtons) required to pass the antibody formulation through the injection needle. The injection force is related to the amount of resistance provided by the antibody formulation when the antibody formulation is administered to a subject. The injection force will depend on the gauge of the administration needle, as well as the temperature. In some embodiments, the antibody formulation has an injection force of less than 15N, 12N, 10N, or 8N when passed through a 27Ga thin tube PFS injection needle. In some embodiments, the antibody formulation has an injection force of less than 15N, 12N, 10N, or 8N when passed through a 29Ga thin tube PFS injection needle.

The antibody formulations may have different osmolarity. Methods of measuring osmolarity of antibody formulations are known to those skilled in the art and may include, for example, an osmometer (e.g., advanced instrumentation 2020 (Advanced instrumentation Inc 2020) freezing point depression osmometer). In some embodiments, the formulation has an osmolarity between 200 and 600mosm/kg, between 260 and 500mosm/kg, or between 300 and 450 mosm/kg.

The antibody formulations of the invention may have different pH levels. In some embodiments, the pH of the antibody formulation is between 4 and 7, between 4.5 and 6.5, or between 5 and 6. In some embodiments, the pH of the antibody formulation is 5.0. In some embodiments, the pH of the antibody formulation is 6.0. In some embodiments, the pH of the antibody formulation is ≧ 7.0. The desired pH level may be achieved by various means including, but not limited to, the addition of an appropriate buffer.

Other different components may be included in the antibody formulation. In some embodiments, the antibody formulation may comprise a buffer (e.g., a histidine, acetate, phosphate, or citrate buffer), a surfactant (e.g., a polysorbate), and/or a stabilizer (e.g., human blood albumin), among others. In some embodiments, the antibody formulation may comprise a pharmaceutically acceptable carrier including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), sucrose, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), silica sol, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polyethylene-polyoxypropylene-block polymers, and polyethylene glycol. In some embodiments, the antibody formulation further comprises a surfactant. In some embodiments, the surfactant is selected from the group consisting of: polysorbate, sodium lauryl sulfate, and a nonionic surfactant.

In some embodiments, the surfactant is polysorbate 20, i.e., polyoxyethylene (20) sorbitan monolaurate, as represented by the following formula:

polysorbate 20 (PS-20) is commercially available from several commercial suppliers, for example

TW20 (oxtex, brazil (Oxiteno, brazil)) and->

20 (Pierce, rockford IL). Applicants have found that by carefully controlling the concentration of PS-20 in the antibody formulation, the antibody has increased stability and reduced amount of particle formation when stored for long periods of time.

In some embodiments, PS-20 is about 0.001% to about 0.02%, about 0.002% to about 0.015%, about 0.002% to about 0.01%, about 0.004% to about 0.009%, about 0.005% to about 0.008%, about 0.007%, or about 0.006% of the antibody formulation.

In some embodiments, the antibody formulation further comprises histidine. In some embodiments, the antibody formulation comprises about 1mM to about 100mM, about 5mM to about 80mM histidine, about 10mM to about 60mM histidine, about 15mM to about 50mM histidine, about 15mM to about 30mM histidine, or about 20mM histidine.

In some embodiments, different components may be omitted from the antibody formulation, or may be "substantially free" of that component. As used herein, the term "substantially free" refers to an antibody formulation containing less than 0.01%, less than 0.001%, less than 0.0005%, less than 0.0003%, or less than 0.0001% of a specified component.

In some embodiments, the antibody formulation is substantially free of saccharide, i.e., the antibody formulation contains less than 0.01%, less than 0.001%, less than 0.0005%, less than 0.0003%, or less than 0.0001% saccharide. As used herein, the term "saccharide" refers to a class of molecules that are derivatives of polyols. Saccharides are often referred to as carbohydrates and may contain varying amounts of saccharide(s) units, such as monosaccharides, disaccharides and polysaccharides. In some embodiments, the formulation is substantially free of disaccharides. In some embodiments, the formulation is substantially free of reducing sugars, non-reducing sugars, or sugar alcohols. In some embodiments, the antibody formulation is substantially free of proline, glutamate, sorbitol, divalent metal ions, and/or succinate.

In some embodiments, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.002% to about 0.01% polysorbate-20; (c) about 40mM to about 60mM trehalose; and (d) about 110mM to about 150mM L-arginine. In some embodiments, the formulation further comprises about 20mM histidine. In one embodiment, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 50mM trehalose; (d) about 130mM L-arginine; and about 20mM histidine.

In some embodiments, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.002% to about 0.01% polysorbate-20; (c) about 200mM to about 300mM trehalose; and (d) about 20mM histidine. In one embodiment, the present invention relates to a stable aqueous antibody formulation comprising: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 250mM trehalose; and (d) about 20mM histidine. In another embodiment, the invention relates to a stable aqueous antibody formulation comprising: (a) About 30mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) about 0.006% polysorbate-20; (c) about 250mM trehalose; and (d) about 20mM histidine.

The antibody formulations of the invention are aqueous solutions. In some embodiments, the antibody formulations have not been subjected to a freezing temperature, and/or have not been frozen, i.e., they remain in a liquid state. In some embodiments, the antibodies in the antibody formulation have not been subjected to lyophilization.

As used herein, the term "stability" generally relates to maintaining the integrity of or minimizing degradation, denaturation, aggregation, or unfolding of a bioactive agent (such as a protein, peptide, or another bioactive macromolecule). As used herein, "improved stability" generally means that a protein of interest (e.g., an antibody such as an anti-IL 5R), a peptide, or another biologically active macromolecule maintains greater stability than a control protein, peptide, or another biologically active macromolecule under conditions known to result in degradation, denaturation, aggregation, or unfolding.

In some embodiments, stability refers to antibody formulations having low to undetectable levels of particle formation. As used herein, the phrase "low to undetectable particle formation level" means that the sample contains less than 30 particles/mL, less than 20 particles/mL, less than 15 particles/mL, less than 10 particles/mL, less than 5 particles/mL, less than 2 particles/mL, or less than 1 particle/mL, as determined by HIAC analysis or visual analysis. In some embodiments, no particles are detected in the antibody formulation, whether by HIAC analysis or visual analysis.

In some embodiments, stability refers to reduced antibody fragmentation. As used herein, the term "low to undetectable fragmentation level" refers to a sample that is equal to or greater than 80%, 85%, 90%, 95%, 98%, or 99% of the total protein within, for example, a single peak as determined by HPSEC, or within two peaks (e.g., heavy and light chains) (or the same number of peaks as the number of subunits) as determined by reduced capillary gel electrophoresis (rCGE), represents a non-degraded antibody or non-degraded fragment, and does not contain additional single peaks each having greater than 5%, greater than 4%, greater than 3%, greater than 2%, greater than 1%, or greater than 0.5% of the total protein. As used herein, the term "reduced capillary gel electrophoresis" refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in antibodies.

One skilled in the art will appreciate that the stability of a protein depends on other properties besides the composition of the formulation. For example, stabilization may be affected by temperature, pressure, humidity, pH, and radiation extrinsic forms. Thus, unless otherwise specified, the stability referred to herein is considered to be measured at 5 ℃, one atmosphere, 50% relative humidity, pH of 6.0, and normal radiation background levels. The stability of the antibody in the antibody formulation can be determined by different means. In some embodiments, antibody stability is determined by Size Exclusion Chromatography (SEC). SEC separates analytes (e.g., macromolecules such as proteins and antibodies) based on their hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate an antibody in its native three-dimensional conformation from an antibody in a different denatured state and/or degraded antibody. In SEC, the stationary phase is usually composed of inert particles packed into a dense three-dimensional matrix inside a glass or steel column. The mobile phase may be pure water, aqueous buffers, organic solvents, mixtures of these, or other solvents. The stationary phase particles have small pores and/or channels that will only allow substances below a certain size to enter. Thus, large particles are excluded from these pores and channels, and smaller particles are removed from the flowing mobile phase. The time it takes for the particles to settle in the stationary phase pores depends in part on the rate at which the particles can penetrate the pores. The removal of the particles from the mobile phase stream causes them to take longer to elute from the column and results in separation between particles based on particle size differences.

In some embodiments, SEC is combined with a discrimination technique to identify or characterize a protein or fragment thereof. Protein identification and characterization can be accomplished by different techniques, including but not limited to chromatographic techniques (e.g., high Performance Liquid Chromatography (HPLC)), immunoassay, electrophoresis, ultraviolet/visible/infrared spectroscopy, raman spectroscopy, surface enhanced raman spectroscopy, mass spectrometry, gas chromatography, static Light Scattering (SLS), fourier transform infrared spectroscopy (FTIR), circular Dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS protein binding.

In some embodiments, protein identification is achieved by high pressure liquid chromatography. Different instruments and equipment for performing HPLC are known to the person skilled in the art. Typically, HPLC involves loading a liquid solvent containing the protein of interest onto a separation column where separation occurs. HPLC separation columns are packed with solid particles (e.g., silica, polymers, or adsorbents), and the sample mixture separates into a variety of compounds upon interaction with the column particles. HPLC separation is subject to conditions of the liquid solvent (e.g., pressure, temperature), chemical interactions between the sample mixture and the liquid solvent (e.g., hydrophobicity, protonation, etc.), and chemical interactions between the sample mixture and solid particles packed within the separation column (e.g., ligand affinity, ion exchange, etc.).

In some embodiments, SEC and protein identification occur in the same device or simultaneously. For example, SEC and HPLC may be combined, often referred to as SE-HPLC.

In some embodiments, the aqueous formulation comprises about 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein said formulation is stable upon storage at about 40 ℃ for at least 1 month. In some embodiments, the formulation is stable upon storage at about 25 ℃ for at least 3 months. In some embodiments, the formulation is stable upon storage at about 5 ℃ for at least 6 months. In some embodiments, the formulation is stable upon storage at about 5 ℃ for at least 12 months. In some embodiments, the formulation is stable upon storage at about 5 ℃ for at least 18 months. In some embodiments, the formulation is stable upon storage at about 5 ℃ for at least 24 months or 36 months.

The term "stable" may be relative and not absolute. Thus, in some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 2 ℃ to 8 ℃ for 6 months. In some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 2 ℃ to 8 ℃ for 12 months. In some embodiments, the antibody in the antibody formulation is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored for 18 months at 2 ℃ to 8 ℃. In some embodiments, an antibody in an antibody formulation is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 2 ℃ to 8 ℃ for 24 months.

In some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 23 ℃ to 27 ℃ for 3 months. In some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 23 ℃ to 27 ℃ for 6 months. In some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 23 ℃ to 27 ℃ for 12 months. In some embodiments, an antibody is stable if less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 23 ℃ to 27 ℃ for 24 months.

In some embodiments, an antibody is stable if less than 6%, less than 4%, less than 3%, less than 2%, or less than 1% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 40 ℃. In some embodiments, an antibody is stable if less than 6%, less than 4%, less than 3%, less than 2%, or less than 1% of the antibody degrades, denatures, aggregates, or unfolds as determined by SEC HPLC when the antibody is stored at 5 ℃.

In some embodiments, an antibody formulation of the invention is considered stable if the antibody exhibits little loss of binding activity of the antibody (including antibody fragments thereof) of the formulation compared to a reference antibody over a period of 8 weeks, 4 months, 6 months, 9 months, 12 months, or 24 months, as measured by an antibody binding assay known to those of skill in the art, e.g., ELISA. In some embodiments, an antibody stored at about 40 ℃ for at least 1 month retains at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of binding ability to an IL-5 receptor polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 5 ℃ for at least 6 months retains at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of binding ability to an IL-5 receptor polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 40 ℃ for at least 1 month retains at least 95% of binding ability to an IL-5 receptor polypeptide compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 5 ℃ for at least 6 months retains at least 95% of binding ability to an IL-5 receptor polypeptide compared to a reference antibody that has not been stored.

The antibody formulation can provide low to undetectable levels of antibody aggregation. As used herein, the phrase "low to undetectable aggregation levels" refers to an antibody that contains no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, and no more than about 0.5% aggregation by weight of the protein as measured by High Performance Size Exclusion Chromatography (HPSEC) or Static Light Scattering (SLS) techniques. In some embodiments, less than 2% of the antibody forms an aggregate after storage at about 40 ℃ for at least 4 weeks, as determined by HPSEC. In some embodiments, less than 2% of the antibody forms an aggregate after storage at about 5 ° for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months, as determined by HPSEC.

Applicants have found that the antibody formulations provided herein allow for a substantial reduction in particle formation as determined by visual inspection, microfluidic imaging (MFI), or Size Exclusion Chromatography (SEC). In some embodiments, the formulation is substantially free of particles after storage at about 40 ℃ for at least 1 month, as determined by visual inspection. In some embodiments, the formulation is substantially free of particles after storage at about 5 ° for at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months, as determined by visual inspection.

In some embodiments, the antibody formulations of the invention may be used for pharmaceutical purposes. Antibodies used in pharmaceutical applications must be of high purity, particularly with respect to contaminants from cell cultures, including cellular protein contaminants, cellular DNA contaminants, viruses, and other infectious agents. See "WHO for the requirements of the use of animal cells as an in vitro matrix in the production of biologicals: requirements for biologically active Substances No.50 "878, appendix 1,1998 (" WHO Requirements for the use of animal cells as in vitro substrates for the production of biologicals: requirements for Biological subsystems No.50."No.878 Annex 1, 1998). In response to concerns about pollutants, the World Health Organization (WHO) sets limits on various levels of pollutants. For example, WHO recommends DNA limits per dose of less than 10ng for protein products. Similarly, the U.S. Food and Drug Administration (FDA) sets a DNA limit to less than or equal to 0.5pg/mg protein. Accordingly, in some embodiments, the invention relates to antibodies that meet or exceed the contaminant limits as defined by one or more governmental agencies, such as the U.S. food and drug administration and/or the world health organization.

In some embodiments, the antibody formulations described herein are pharmaceutically acceptable. "pharmaceutically acceptable" refers to antibody formulations which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, or other complications commensurate with a reasonable benefit/risk ratio.

The purity of the antibody formulation may vary. In some embodiments, the therapeutic antibody of interest (e.g., an anti-IL 5R antibody) is greater than 90% (weight/weight) of the total polypeptide present in the antibody formulation. In some embodiments, the therapeutic antibody of interest (e.g., anti-IL 5R) is greater than 95% (weight/weight), 98% (weight/weight), 99% (weight/weight), 99.5% (weight/weight), 99.9% (weight/weight) of the total polypeptide present in the antibody formulation.

The formulations provided herein can be suitable for treating a subject. As used herein, "subject" is used interchangeably with "patient" and refers to any animal classified as a mammal, including humans and non-humans, such as, but not limited to, livestock and farm animals, zoo animals, sports animals, and pets. In some embodiments, the subject is a human.

The terms "treatment" and "treatment" refer to both therapeutic treatment and prophylactic, maintenance, or preventative measures, wherein the object is to prevent or slow down (lessen) an undesirable physiological condition, disorder or disease, or to achieve a beneficial or desired clinical result. The terms "treat," "treatment," and "treating" refer to reducing or ameliorating the progression, severity, and/or duration of such a disease or disorder (e.g., a disease or disorder characterized by aberrant expression and/or activity of an IL-5 polypeptide or one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease or infection), or ameliorating one or more symptoms of such a disease or disorder, by administering one or more therapies, including but not limited to administration of one or more prophylactic or therapeutic agents. In certain embodiments, such terms refer to the reduction of inflammation-associated eosinophil-mediated inflammation. In other embodiments, such terms refer to a reduction in inflammatory factors released by mast cells, or a reduction in the biological effects of such inflammatory factors. In other embodiments, such terms refer to a reduction in the growth, formation, and/or increase in the number of hyperproliferative cells (e.g., cancerous cells). In yet other embodiments, such terms refer to a reduction in inflammation of the airway, skin, gastrointestinal tract, or a combination thereof. In yet other embodiments, such terms refer to a reduction in symptoms associated with asthma. In yet other embodiments, such terms refer to a reduction in symptoms associated with Chronic Obstructive Pulmonary Disease (COPD).

The antibody formulations of the invention may be administered to a subject by different means. In some embodiments, the antibody formulation is suitable for parenteral administration, e.g., via inhalation (e.g., powder or spray), transmucosal, intravenous, subcutaneous, or intramuscular administration. In some embodiments, the formulation is an injectable formulation. In some embodiments, the invention relates to a sealed container comprising any antibody formulation as described herein.

In some aspects, the invention relates to different pharmaceutical dosage forms. Different dosage forms may be applied to the formulations provided herein. See, e.g., pharmaceutical dosage forms: parenteral drugs, vol.1, 2nd edition (Pharmaceutical Dosage Form: parenteral medicines,

volume

1,2 ^nd Edition). In one embodiment, the pharmaceutical unit dosage form of the invention comprises the antibody formulation in a suitable container (e.g., vial or syringe). In one embodiment, the pharmaceutical unit dosage form of the invention comprises an intravenously, subcutaneously, or intramuscularly delivered antibody formulation. In another embodiment, the pharmaceutical unit dosage form of the invention comprises an aerosol-delivered antibody formulation. In particular embodiments, the pharmaceutical unit dosage forms of the invention comprise a subcutaneously delivered antibody formulation. In another embodiment, the pharmaceutical unit dosage form of the invention comprises an aerosol-delivered antibody formulation. In further embodiments, the pharmaceutical unit dosage forms of the invention comprise an intranasally administered antibody formulation.

The antibody formulations of the invention can be prepared by preparing a vial containing an aliquot of the aqueous antibody formulation for single use as a unit dosage form. For example, a unit dose per vial may contain varying concentrations of 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 15ml, or 20ml of an antibody that specifically binds to the IL5 receptor, ranging from about 0.1mg/ml to about 300mg/ml. These formulations can be adjusted to the desired concentration by adding a sterile diluent to each vial, if necessary. In particular embodiments, the aqueous antibody formulations of the present invention are formulated into single dose vials as a sterile liquid containing: about 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; about 0.002% to about 0.01% polysorbate-20; about 40mM to about 60mM trehalose; and about 110mM to about 150mM L-arginine. In another particular embodiment, the aqueous antibody formulations of the present invention are formulated into single dose vials as a sterile liquid containing: about 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; about 0.002% to about 0.01% polysorbate-20; and about 200mM to about 300mM trehalose. In one embodiment, the antibodies of the invention are provided at 2 to 20mg/ml in 3cc USP type I borosilicate amber vials (West Pharmaceutical Services-Part No. 6800-0675)). In one embodiment, the antibodies of the invention are provided at 20 to 100mg/ml in 3cc USP type I borosilicate amber vials. The target fill volume was 1.2mL.

The antibody formulations of the invention may be prepared by preparing a pre-filled syringe containing an aliquot of the aqueous antibody formulation for single use as a unit dosage form. For example, a unit dose per pre-filled syringe may contain varying concentrations of antibody that specifically binds to an IL-5 polypeptide ranging from about 2mg/ml to about 100mg/ml at 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml, 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 15ml, or 20 ml. In particular embodiments, the aqueous antibody formulations of the present invention are formulated into single-dose pre-filled syringes as a sterile liquid containing: about 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; about 0.002% to about 0.01% polysorbate-20; about 40mM to about 60mM trehalose; and about 110mM to about 150mM L-arginine. In particular embodiments, the aqueous antibody formulations of the present invention are formulated into single-dose pre-filled syringes as a sterile liquid containing: about 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; about 0.002% to about 0.01% polysorbate-20; and about 200mM to about 300mM trehalose.

Different dosage amounts may be administered in a single use. For example, in some embodiments, 0.1mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1.0mg, 1.1mg, 1.2mg, 1.3mg, 1.4mg, 1.5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 12mg, 14mg, 16mg, 18mg, 20mg, 30mg, 40mg, 50mg, 70mg, or 100mg of the antibody can be administered in a single dose.

Different types of syringes may be used. The syringe can be filled with the antibody formulation immediately prior to administration to the subject, e.g., less than 1 week, 1 day, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes prior to administration to the subject. In some embodiments, the syringe may be filled with the antibody formulation at a retail point of sale, or by the facility where the subject is treated. In some embodiments, the syringe is prefilled, e.g., filled with the antibody formulation, more than 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, 24 months, 3 years, or 4 years prior to administration to the subject. In some embodiments, the pre-filled syringe comprises an injection needle, such as a 27G conventional thin-tube injection needle, a 27G thin-tube injection needle, a 29G conventional thin-tube injection needle, or a 29G thin-tube injection needle. In some embodiments, the pre-filled syringe comprises a 29G thin-tube injection needle.

In some embodiments, any syringe suitable for administration to a desired subject may be used. In some embodiments, the syringe is a plastic syringe or a glass syringe. In some embodiments, the injector is made of a material that is substantially free of tungsten. In some embodiments, the syringe is coated with silicone. In some embodiments, the pre-filled syringe includes a plunger having a fluoropolymer resin disk. Examples of syringes may include, but are not limited to, 1ml long Hypak ^TM for Biotech (Becton Dickinson) with a 1mL long Becton Dickinson Hypak plunger stopper 4023Flurotec Daikyo Si1000 (Cat. No. 47271919), C3Pin (Lot. No. E912701); hypak of 0.8mg silicone oil ^TM for Biotech (Bidi Corp.); and CZ syringes (West corporation, catalog No. 19550807).

The aqueous antibody formulations of the present invention may be sterilized by various sterilization methods, including sterile filtration, irradiation, and the like. In particular embodiments, the diafiltered antibody formulation is sterile filtered with a presterilized 0.2 micron filter. The sterilized aqueous antibody formulations of the present invention can be administered to a subject to prevent, treat and/or manage an immune response, such as an inflammatory response.

In some embodiments, the pre-filled syringe comprises: (a) About 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) about 0.002% to about 0.01% polysorbate-20. In some embodiments, the pre-filled syringe further comprises: (c) About 40mM to about 60mM trehalose, and (d) about 110mM to about 150mM L-arginine. In some embodiments, the pre-filled syringe comprises: (a) About 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) about 0.002% to about 0.01% polysorbate-20. In some embodiments, the pre-filled syringe further comprises: (c) about 200mM to about 300mM trehalose.

In some embodiments, the invention relates to a kit comprising any of the antibody formulations described herein, the containers described herein, the unit dosage forms described herein, or the pre-filled syringes described herein.

In some embodiments, the present invention may also relate to a method of preparing a stable aqueous antibody formulation comprising an antibody, the method comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (b) placing the isolated antibody in a stabilizing formulation to form the stable aqueous antibody formulation, wherein the resulting stable aqueous antibody formulation comprises: (i) About 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (ii) about 0.002% to about 0.01% polysorbate-20. In some embodiments, the present invention relates to a method of preparing a stable aqueous antibody formulation comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) Diluting the antibody to about 2mg/mL to about 20mg/mL of antibody in a solution comprising: (i) about 0.002% to about 0.01% polysorbate-20; (ii) about 40mM to about 60mM trehalose; and (iii) about 110mM to about 150mM L-arginine, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 8-10. In some embodiments, the present invention relates to a method of preparing a stable aqueous antibody formulation comprising: (a) Purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; (b) Diluting the antibody to about 20mg/mL to about 100mg/mL of antibody in a solution comprising: (i) about 0.002% to about 0.01% polysorbate-20; (ii) about 200mM to about 300mM trehalose; and (iii) about 20mM histidine, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 8-10.

Although many aspects of the invention relate to aqueous formulations, it should be noted that for equivalent purposes, the antibodies or antibody formulations of the invention may be lyophilized, if desired. Thus, the invention encompasses the formulation of the invention in lyophilized form, or lyophilized antibody which is later reconstituted to aqueous form. In some embodiments, the present invention relates to a method of making a reconstituted antibody formulation comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, the method comprising: (ii) (a) purifying the antibody from the cell culture; (b) lyophilizing the isolated antibody; (c) Adding the lyophilized antibody to an aqueous solution to form a reconstituted antibody formulation, wherein the reconstituted antibody formulation comprises: (i) About 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and (ii) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the inventors have found that anti-IL 5R antibody formulations with increased glutathione S-transferase (GST) cause reduced (e.g., undetectable) particle formation. Removal of particles is important to avoid potential immunogenicity and to limit the impact on product quality. In some embodiments, the GST concentration is reduced by affinity chromatography. In some embodiments, the GST concentration is reduced by using a protein a column. In some embodiments, the protein a column is MabSelect Sure (GE medical Life Sciences). In some embodiments, the GST concentration is reduced by using mixed mode chromatography. In some embodiments, the mixed-mode column is Capto ^TM Adhere (GE medical Life sciences).

In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of particles. In some embodiments, the term "substantially free of particles" means that there are no visible particles when viewed under a light box. In some embodiments, the term "substantially free of particles" is synonymous with the phrase "low to undetectable particle formation level" as previously described. In some embodiments, substantially free of particles means that the sample contains less than 30 particles/mL, less than 20 particles/mL, less than 15 particles/mL, less than 10 particles/mL, less than 5 particles/mL, less than 2 particles/mL, or less than 1 particle/mL, wherein the particles are greater than 25 μm and the particle count is determined by HIAC analysis or visual analysis. In some embodiments, substantially free of particles means that the sample contains 1 to 50 particles/mL, 2 to 40 particles/mL, 3-30 particles/mL, 4 to 25 particles/mL, or 5 to 20 particles/mL, wherein the particles are greater than 25 μm and the particle count is determined by HIAC analysis or visual analysis. In some embodiments, the term "visible particle" refers to a particle greater than 25 μm.

In some embodiments, substantially free of particles means that the sample contains 1 to 200 particles/mL, 10 to 150 particles/mL, 30-100 particles/mL, or 40 to 80 particles/mL, wherein the particles are greater than 5 μm and the particle count is determined by HIAC analysis or visual analysis. In some embodiments, the term "visible particle" refers to a particle greater than 5 μm. In some embodiments, no particles are detected in the antibody formulation, whether by HIAC analysis or visual analysis.

In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of glutathione S-transferase (GST). Unless otherwise indicated, the terms "substantially free of glutathione S-transferase" or "substantially free of GST" shall encompass compositions lacking active GST (but may contain inactive GST) as well as compositions without GST protein (whether in active or inactive form). In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of active GST. The term "active GST" refers to GST capable of catalyzing the formation of a thiol group of Glutathione (GSH) to an electrophilic compound such as 1-chloro-2, 4-dinitrobenzene (CDNB) to form a GS-DNB conjugate. GST or glutathione S-transterTransferases refer to a family of enzymes: it is capable of catalyzing a variety of reactions, but primarily reduced glutathione is conjugated to the electrophilic center via sulfhydryl groups (e.g., aromatics, double bonds, C-Cl) _x Etc.). GST monomers are in the general range of 22-29kDa and they can occur as dimers, trimers and also heterodimers (with other proteins). In some embodiments, the term GST refers to a protein capable of catalyzing the formation of the thiol group of Glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene (CDNB) so as to form a GS-DNB conjugate.

In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of particles when stored at 38 ℃ -42 ℃ for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, or at least 18 months. In some embodiments, the invention relates to an antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of particles when stored at 2 ℃ -6 ℃ for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or at least 48 months.

In some embodiments, the antibody formulation is substantially free of GST. In some embodiments, the term "substantially free of GST" refers to an antibody formulation having GST activity less than about 0.5 units/mg antibody, less than about 0.3 units/mg antibody, less than about 0.1 units/mg antibody, less than about 0.08 units/mg antibodyLess than about 0.05 units/mg antibody, less than about 0.03 units/mg antibody, less than about 0.01 units/mg antibody, less than about 0.005 units/mg antibody, less than about 0.001 units/mg antibody, less than about 5X 10 ^-3 Less than about 1X 10 units/mg antibody ^-4 Less than about 1X 10 units/mg antibody ^-5 Units/mg antibody, or less than about 11X 10 ^-6 Units/mg antibody. In some embodiments, the term "substantially free" means that the level of GST is not detectable using common GST detection techniques.

Different methods for determining GST activity are known to those skilled in the art. In some embodiments, GST activity is determined using a glutathione (GSH/GSSG/total) fluorescence assay kit (bio field, san Francisco CA).

In some embodiments, the invention relates to a method of purifying an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 8-10, the method comprising: (i) obtaining a cell culture comprising the antibody; (ii) performing affinity chromatography on the antibody; (iv) performing cation exchange on the antibody; (v) performing mixed mode chromatography on the antibody. In some embodiments, the invention relates to a method of purifying an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, the method comprising: (i) obtaining a cell culture comprising the antibody; (ii) binding the antibody to a protein a column; (iii) eluting the antibody from the protein a column; (iv) performing cation exchange on the antibody; (v) performing mixed mode chromatography on the antibody. In some embodiments, the method of purifying an antibody further comprises a viral inactivation process. In some embodiments, the virus inactivation step is performed by lowering the pH to less than 4.0. In some embodiments, the method further comprises a diafiltration process. In some embodiments, the method further comprises a filtration process. In some embodiments, the filtration process is sufficient to remove active virus particles.

In some embodiments, the invention relates to a method of treating a patient. In some embodiments, the invention comprises administering to a subject in need thereof an antibody formulation described herein, a container described herein, a unit dosage form described herein, or a pre-filled syringe described herein.

In some embodiments, the invention is suitable for treating a pulmonary disease or disorder by administering the antibody formulations described herein. In some embodiments, the invention relates to a method of treating a patient having an eosinophilic disease or disorder by administering an antibody formulation described herein. In some embodiments, the invention relates to a method of treating a pulmonary disease or disorder in a subject, the method comprising administering an antibody formulation described herein. In some embodiments, the invention relates to a method of treating an eosinophilic disease or disorder in a subject comprising administering an antibody formulation described herein. In some embodiments, the present invention relates to the treatment of the following pulmonary diseases or disorders in a subject: for example, asthma, COPD, eosinophilic asthma, eosinophilic and neutrophilic co-asthma, aspirin-sensitive asthma, allergic bronchopulmonary aspergillosis, acute and chronic eosinophilic bronchitis, acute and chronic eosinophilic pneumonia, churg-schii syndrome, hyper-eosinophilic syndrome, drug, irritant and radiation-induced hypereosinophilia, infection-induced hypereosinophilia (fungi, tuberculosis, parasites), autoimmune-related hypereosinophilia, eosinophilic esophagitis, or crohn's disease, or a combination thereof, comprising administering the antibody formulations described herein. In some embodiments, the invention relates to the treatment of asthma in a subject, the method comprising administering an antibody formulation described herein. In some embodiments, the invention relates to the treatment of COPD in a subject comprising administering an antibody formulation described herein.

In some embodiments, a therapeutically effective amount of an antibody formulation described herein is administered to treat a condition. As used herein, the term "therapeutically effective amount" refers to an amount of a therapy (e.g., an antibody that immunospecifically binds to an IL-5 receptor polypeptide) sufficient to reduce the severity of a disease or disorder (e.g., a disease or disorder characterized by aberrant expression and/or activity of an IL-5 polypeptide or one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease or infection (preferably a respiratory infection), or one or more symptoms thereof), reduce the duration of a respiratory disorder, ameliorate one or more symptoms of such a disease or disorder, prevent progression of such a disease or disorder, cause regression of such a disease or disorder, or enhance or improve the therapeutic effect of another therapy. In some embodiments, the therapeutically effective amount cannot be specified in advance, and can be determined by a caregiver (e.g., by a physician or other healthcare provider) using various means (e.g., dose adjustment). An appropriate therapeutically effective amount can also be determined by routine experimentation using, for example, animal models.

The terms "therapy (" therapies "and" therapy ")" may refer to any regimen, method, and/or agent that can be used to prevent, treat, manage, or ameliorate a disease or disorder (e.g., a disease or disorder characterized by aberrant expression and/or activity of an IL-5 polypeptide or one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease or infection (preferably, a respiratory infection), or one or more symptoms thereof). In certain embodiments, the term "therapy (" therapies "and" therapy ") refers to biological therapy, supportive therapy, and/or other therapies known to skilled medical personnel to be useful in treating, managing, preventing, or ameliorating such a disease or disorder or one or more symptoms.

As used herein, the term "treatment regimen" refers to a regimen of dosages and times for scheduling the administration of one or more therapies (e.g., therapeutic agents) having a therapeutic effect.

The route of administration of the antibody formulations of the invention may be, for example, via oral, parenteral, inhalation, or topical modes of administration. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. In some embodiments, the antibody is an anti-IL 5R antibody and the route of administration is intramuscular injection. Although all of these administration forms are clearly contemplated as being within the scope of the present invention, in some embodiments, the antibody formulation is suitable for administration via injection, particularly via intravenous or intra-arterial injection or instillation.

In some embodiments, the compositions and methods of the invention enable manufacturers to produce antibody formulations suitable for administration to humans in a more efficient manner, reducing cost, reducing process steps, reducing the chance of error, reducing the chance of unsafe or inappropriate additive introduction, reducing waste, increasing storage time, and the like.

Detailed Description

The present invention also includes the following embodiments:

1. a stable aqueous antibody formulation comprising:

a. about 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and

b. about 0.002% to about 0.01% polysorbate-20.

2. The antibody formulation of embodiment 1, further comprising an uncharged excipient.

3. The antibody formulation of embodiment 2, wherein the uncharged excipient is trehalose.

4. The antibody formulation of any one of embodiments 1 to 3, comprising about 2 to about 20mg/ml of the antibody.

5. The antibody formulation of any one of embodiments 1 to 3, comprising about 20 to about 100mg/ml of the antibody.

6. The antibody formulation of embodiment 4, wherein the concentration of the uncharged excipient is about 20mM to about 80mM.

7. The antibody formulation of embodiment 5, wherein the concentration of the uncharged excipient is about 200mM to about 400mM.

8. The antibody formulation of embodiment 4, further comprising arginine.

9. The antibody formulation of embodiment 8, wherein the arginine is L-arginine.

10. The antibody formulation of embodiment 8, comprising about 100mM to about 200mM L-arginine.

11. The antibody formulation of embodiment 8, comprising about 120mM to about 140mM L-arginine, and about 40mM to about 60mM uncharged excipient.

12. The antibody formulation of any one of embodiments 1 to 11, further comprising histidine.

13. The antibody formulation of embodiment 12, wherein the concentration of histidine is about 15mM to about 30mM.

14. The antibody formulation of any one of embodiments 1 to 13, wherein said antibody is not subjected to lyophilization.

15. A stable aqueous antibody formulation comprising about 2mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein said formulation is stable upon storage at about 40 ℃ for at least 1 month.

16. A stable aqueous antibody formulation comprising:

a. about 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10;

b. about 0.002% to about 0.01% polysorbate-20;

c. about 40mM to about 60mM trehalose;

d. about 110mM to about 150mM L-arginine; and

e. about 15mM to about 30mM histidine.

17. A stable aqueous antibody formulation comprising:

a. about 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10;

b. about 0.002% to about 0.01% polysorbate-20;

c. about 200mM to about 300mM trehalose; and

d. about 15mM to about 30mM histidine.

18. A stable aqueous antibody formulation comprising:

b. about 0.006% polysorbate-20;

c. about 50mM trehalose;

d. about 130mM L-arginine; and

e. about 20mM histidine.

19. A stable aqueous antibody formulation comprising:

b. about 0.006% polysorbate-20;

c. about 250mM trehalose; and

d. about 20mM histidine.

20. A stable aqueous antibody formulation comprising:

a. about 30mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10;

b. about 0.006% polysorbate-20;

c. about 250mM trehalose; and

d. about 20mM histidine.

21. The antibody formulation of any one of embodiments 1 to 20, wherein the formulation is stable upon storage at about 25 ℃ for at least 3 months.

22. The antibody formulation of any one of embodiments 1 to 21, wherein the formulation is stable upon storage at about 5 ℃ for at least 18 months.

23. The antibody formulation of any one of embodiments 1 to 22, wherein the antibody stored at about 40 ℃ for at least 1 month retains at least 80% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored.

24. The antibody formulation of any one of embodiments 1 to 23, wherein the antibody stored at about 5 ℃ for at least 6 months retains at least 80% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored.

25. The antibody formulation of any one of embodiments 1 to 24, wherein the antibody stored at about 40 ℃ for at least 1 month retains at least 95% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored.

26. The antibody formulation of any one of embodiments 1 to 25, wherein the antibody stored at about 5 ℃ for at least 6 months retains at least 95% of binding ability to an IL-5R polypeptide compared to a reference antibody that has not been stored.

27. The antibody formulation of any one of embodiments 1 to 26, wherein less than 2% of the antibody forms an aggregate upon storage at about 40 ℃ for at least 1 month as determined by HPSEC.

28. The antibody formulation of any one of embodiments 1 to 27, wherein less than 2% of the antibody forms an aggregate upon storage at about 5 ℃ for at least 12 months as determined by HPSEC.

29. The antibody formulation of any one of embodiments 1 to 28, wherein the formulation is substantially free of particles after storage at about 40 ℃ for at least 1 month as determined by visual inspection.

30. The antibody formulation of any one of embodiments 1 to 29, wherein the formulation is substantially free of particles after storage at about 5 ℃ for at least 12 months as determined by visual inspection.

31. The antibody formulation of any one of embodiments 1 to 30, wherein the formulation is an injectable formulation.

32. The antibody formulation of any one of embodiments 1 to 31, wherein the formulation is suitable for intravenous, subcutaneous, or intramuscular administration.

33. A sealed container containing an antibody formulation according to any one of embodiments 1 to 32.

34. A pharmaceutical unit dosage form suitable for parenteral administration to a human comprising the antibody formulation of any one of embodiments 1 to 32 in a suitable container.

35. The pharmaceutical unit dosage form of embodiment 34, wherein the antibody formulation is administered intravenously, subcutaneously, or intramuscularly.

36. The pharmaceutical unit dosage form of embodiment 34 or 35, wherein the suitable container is a pre-filled syringe.

37. The pharmaceutical unit dosage form of embodiment 36, wherein the pre-filled syringe comprises an injection needle.

38. The pharmaceutical unit dosage form of embodiment 37, wherein the injection needle is a 29G thin-tube injection needle.

39. The pharmaceutical unit dosage form of any one of embodiments 36 to 38, wherein the prefilled syringe is a plastic syringe or a glass syringe.

40. The pharmaceutical unit dosage form of any of embodiments 36 to 39, wherein the pre-filled syringe is made of a material that is substantially free of tungsten.

41. The pharmaceutical unit dosage form of any one of embodiments 361 to 40, wherein the pre-filled syringe is silicone coated.

42. The pharmaceutical unit dosage form of any one of embodiments 36 to 41, wherein the pre-filled syringe comprises a plunger having a disk of fluoropolymer resin.

43. A pre-filled syringe comprising:

a. about 2mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and

b. about 0.002% to about 0.01% polysorbate-20.

44. The pre-filled syringe of embodiment 38, further comprising:

c. about 40mM to about 60mM trehalose; and

d. about 110mM to about 150mM L-arginine.

45. A pre-filled syringe comprising:

a. about 20mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises the amino acid sequence of SEQ ID NO 1,

b. about 0.002% to about 0.01% polysorbate-20.

46. The pre-filled syringe of embodiment 40, further comprising:

c. about 200mM to about 300mM trehalose.

47. A pre-filled syringe comprising:

b. polysorbate-20 at about 0.006%.

48. The pre-filled syringe of embodiment 47, further comprising:

c. about 50mM trehalose; and

d. about 130mM L-arginine.

49. A pre-filled syringe comprising:

b. polysorbate-20 at about 0.006%.

50. The pre-filled syringe of embodiment 49, further comprising:

c. about 250mM trehalose.

51. A pre-filled syringe comprising:

a. about 30mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and

b. polysorbate-20 at about 0.006%.

52. The pre-filled syringe of embodiment 51, further comprising:

c. about 250mM trehalose.

53. A kit comprising the formulation of any one of embodiments 1 to 32, the container of embodiment 33, the unit dosage form of any one of embodiments 34 to 42, or the pre-filled syringe of any one of embodiments 43 to 52.

54. A method of making a stable aqueous antibody formulation, the method comprising:

a. purifying an antibody to about 1mg/mL to about 400mg/mL, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by kabat of SEQ ID NOs 8-10;

b. placing the isolated antibody in a stabilizing formulation to form the stable aqueous antibody formulation, wherein the resulting stable aqueous antibody formulation comprises:

i. about 2mg/mL to about 100mg/mL of the antibody; and

from about 0.002% to about 0.01% polysorbate-20.

55. A method of preparing a stable aqueous antibody formulation, the method comprising:

b. diluting the antibody to about 2mg/mL to about 20mg/mL of the antibody in a solution comprising:

i. about 0.002% to about 0.01% polysorbate-20;

from about 40mM to about 60mM trehalose; and

from about 110mM to about 150mM L-arginine.

56. A method of preparing a stable aqueous antibody formulation, the method comprising:

b. diluting the antibody to about 20mg/mL to about 100mg/mL of the antibody in a solution comprising:

i. about 0.002% to about 0.01% polysorbate-20; and

from about 200mM to about 300mM trehalose.

57. A method of making a reconstituted antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, the method comprising:

a. purifying the antibody from the cell culture;

b. lyophilizing the isolated antibody;

c. adding the lyophilized antibody to an aqueous solution to form a reconstituted antibody formulation, wherein the reconstituted antibody formulation comprises:

i. about 2mg/mL to about 100mg/mL of the antibody; and

from about 0.002% to about 0.01% polysorbate-20.

58. An antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of particles.

59. An antibody formulation comprising an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 8-10, wherein the antibody formulation is substantially free of active glutathione S-transferase (GST).

60. The antibody formulation of embodiment 59, wherein the antibody sequence is substantially free of GST.

61. The antibody formulation of embodiment 58, wherein the antibody formulation is substantially free of particles when stored at 38 ℃ -42 ℃ for at least 1 month.

62. The antibody formulation of embodiment 58, wherein the antibody formulation is substantially free of particles when stored at 2 ℃ -6 ℃ for at least 6 months.

63. A method of purifying an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 5-7, and wherein the light chain variable region comprises the kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs 8-10, the method comprising:

a. obtaining a cell culture comprising the antibody;

b. performing affinity chromatography on the antibody;

c. performing cation exchange on the antibody;

d. mixed mode chromatography was performed on the antibody.

64. The method of embodiment 63, further comprising a viral inactivation process.

65. The method of embodiment 63, further comprising a diafiltration process.

66. A method of treating a pulmonary disease or disorder in a subject, the method comprising administering a therapeutically effective amount of the antibody formulation of any one of embodiments 1 to 32, the container of embodiment 33, the unit dosage form of any one of embodiments 34 to 42, or the pre-filled syringe of any one of embodiments 43 to 52.

67. The method of embodiment 49, wherein the pulmonary disease or disorder is an eosinophilic disease or disorder.

68. The method of embodiment 49, wherein the pulmonary disease or disorder is asthma, COPD, eosinophilic asthma, eosinophilic and neutrophilic asthma, aspirin-sensitive asthma, allergic bronchopulmonary aspergillosis, acute and chronic eosinophilic bronchitis, acute and chronic eosinophilic pneumonia, churg-schin-shidi syndrome, hyper-eosinophilic syndrome, a drug, irritant and radiation-induced hypereosinophilia, infection-induced hypereosinophilia (fungal, tuberculosis, parasite), autoimmune-related hypereosinophilia, eosinophilic esophagitis, crohn's disease, or a combination thereof.

69. The method of embodiment 49, wherein the pulmonary disease or disorder is asthma.

70. The method of embodiment 49, wherein the pulmonary disease or disorder is chronic obstructive pulmonary disease.

Examples of the invention

Example 1

Formulation studies were performed to form anti-IL 5R antibody formulations suitable for delivery from pre-filled syringes via subcutaneous delivery (or via a ready-to-use configuration) at doses of 2-100 mg. Specifically, two separate antibody concentration formulations were formed, 2-20mg/mL and 20-100 mg/mL.

1. Materials and methods

a. anti-IL 5R sources and formulation preparation

Multiple batches of anti-IL 5R were used in these studies. All these batches were produced on different scales by MedImmune and delivered after diafiltration and concentration to a concentration of approximately 130g/L in 20mM histidine/histidine hydrochloride at pH 6. Some batches also had 250mM trehalose in the diafiltration buffer.

anti-IL 5R is also by mixing in Excipient Added Buffer (EAB) to the preparation, to achieve 100g/L anti IL5R concentration and buffer and excipient material appropriate concentration. Lower concentrations of drug were prepared from 100g/L formulation.

2. Method for accelerating stress

a. Storage at increased temperature

Vials and syringes are stored in controlled stability chambers to maintain a constant temperature during storage. Maintaining the chamber at 2 ℃ -8 ℃, 23 ℃ -27 ℃/60% rh, or 38 ℃ -42 ℃/75% rh, although their mid-point temperature of 5 ℃,25 ℃ or 40 ℃ will be mentioned below. Unless otherwise indicated, the vials were stored upright and the pre-filled syringe tips were stored down.

b. Freezing and thawing cycle

Both controlled and uncontrolled freeze-thaw cycles were used in these studies. Uncontrolled freeze-thawing was accomplished by freezing the vials in a-40 ℃ chamber and thawing at room temperature.

c. Transportation and rocking

Various methods were used to investigate the effect of transport on anti-IL 5R. The vial was table shaken by shaking at 150rpm for 24 hours. The actual transportation is simulated by shipping the product to an off-site location. The product undergoes two trips and ground and air transport within 4 days. A combination of freezer and ice bags is used to maintain the product temperature at 2-8 ℃, and is monitored by a sensor that indicates a temperature below 0 ℃ or above 9 ℃.

For the screening study, the transport was simulated using a shaking table (transport simulator). The mode in which a product undergoes "air" transport and "ground" transport is the same as that experienced during shuttle. This process took 12 hours and the temperature was again controlled at 2-8 ℃ with ice packs; no sensor is used. The horizontal orientation is chosen as the worst orientation during actual or simulated transport due to the possibility of air bubble travel and the possibility of the medication coming into contact with the entire barrel, needle and stopper.

3. Experimental methods

a. Methods of following or deriving SOP

Visual inspection was performed by comparison to particle and opalescence standards. Aggregation and fragmentation were monitored by SE-HPLC. For concentrations below 10g/L of anti-IL 5R, using larger volume injection to achieve similar per injection total protein mass. Some samples were also used for cIEF measurements and RP-HPLC to monitor fragmentation.

b. Protein concentration

Protein concentration was measured by diluting the protein to approximately 0.5g/L by serial weight dilution and measuring absorbance at 280 nm. Concentrations were calculated from the attenuation coefficient and dilution factor and the effect of density on gravimetric dilution was corrected for initial concentrations above 50 g/L.

c. Sub-visibility particle counting

Sub-visibility particle counts were made using MFI and HIAC. For MFI, after running daylight illumination with water, 0.9mL of solution was run neat. The first 0.2mL was used to purge the system and was not included in the analysis. Spherical bubbles or silicone oil droplets were removed using an aspect ratio filter < 0.85. For HIAC, solutions with concentrations >5g/L were diluted to approximately 5g/L while running pure diluted samples. Dilution was performed in a laminar flow clean bench with 20mM histidine/histidine hydrochloride pH 6 buffer, and filtration at the point of use. The samples were degassed under vacuum for at least 30 minutes prior to testing. The average of three runs was multiplied by the dilution factor to obtain the final result. The silicone oil droplets were not distinguished from the protein particles by HIAC.

4. Data and discussion

a. Polysorbate-20 concentration Screen

The first objective was to optimize the PS-20 concentration in the aqueous antibody formulation. Polysorbates are included in solution to prevent protein denaturation and aggregation at the interface, and the desired concentrations in liquid and lyophilized products are expected to be different. The major interfacial stresses occur during freeze thawing and transport, so the experimental plan focuses on mimicking these. Previous experiments showed that 0.02% polysorbate 20 was sufficient to avoid these stresses completely (data not shown). As a validation, these stresses were combined in series in the order expected for clinical production, and the incubation period was lengthened after the stress to enhance the growth of any protein particles. Most preferably, the drug is subjected to three uncontrolled freeze-thaw cycles, and the material is filtered and filled into vials. It was then shaken on a shaking table and incubated at 40 ℃ for one week before testing by SE-HPLC and MFI. The test conditions were the limits of the concentration range of 2 and 100g/L, formulated in PS-20 at 240mM trehalose, 20mM histidine/histidine hydrochloride (pH 6), and levels varying from 0 to 0.03% w/v. The results are shown in figures 2, 3 and 4.

The monomer fraction data show that the 2g/L solution remains pure regardless of polysorbate level, but a small amount of polysorbate-20 (below about 0.005%) results in aggregation of a small amount of 100g/L, but these results are not indicative of a "failure margin" per se. The stress utilized is severe and the level of degradation is minimal, so any PS-20 level tested will be sufficient from a SEC aggregate perspective. At 2g/L, the sub-visible particle count was quite high and was not controlled by polysorbate-20 in the tested range. At 100g/L, the high sub-visibility particle count is controlled by the presence of 0.003% or more of PS-20. Overall, the data indicate that PS-20 levels should be maintained at 0.003% or above 0.003%.

For low concentration solutions, alternative methods of controlling sub-visible particles are needed, as discussed below.

Screening of pH

The effect of solution pH was studied in 2, 20 and 100g/L solutions, where the pH was between 5 and 7.5. The remainder of the formulation was constant and comprised 240mM trehalose, 20mM histidine/histidine hydrochloride and 0.02% PS-20. The solutions were prepared and stored at 40 ℃ for at least one month before testing. For all samples, monomer loss as determined by SE-HPLC, sub-visible particles as determined by MFI, and visible particles as found by visual inspection were evaluated. Additional tests were performed on 100g/L samples, including RP-HPLC and cIEF. The results are provided in tables 5 and 6.

Aggregation and fragmentation are minimized in the pH range of 5.5 to 6.5. The results from the cIEF are consistent with the reference standard at pH 7.0 and above. The sub-visible particle count was neither low nor showed any pattern with the pH of the solution. Higher particle fractions were seen at pH 5.5-6.5. Even though these fractions were high, these samples were examined to be close to the light at which higher particle counts were routinely seen. It is likely that the source material also resulted in high particle levels, as the HCP level of this material was reduced by further purification on protein a. This study shows that the optimum pH from a particle formation point of view will be pH 5 or pH ≧ 7. However, since the visible particle fraction is understood to be estimated too high here, the pH did not change from pH 6.0 from this study.

c. Conveying influence

The formulations need to be robust to withstand shipping, so tests were performed in pre-filled syringes at different protein and PS-20 concentrations. Formulation changes were 2-100g/L anti-IL 5R and 0-0.05% PS-20, other conditions were constant, i.e., 240mM trehalose, 20mM histidine/histidine hydrochloride, pH 6. A number of other conditions were tested at 2g/L, including glycine, calcium chloride, pH 5.5, pH 6.5, 0.02% polysorbate-80. None of these conditions showed an improvement over trehalose formulations with pH 6 of polysorbate-20 and therefore are not discussed further below.

1mL of the sample was filled into a platform PFS with 0.4mg of silicone oil. The samples were shipped, stored at 5 ℃,25 ℃ and 40 ℃, and tested by visual inspection, MFI and HIAC within two months. The results are presented in fig. 7.

The graph shows the sub-visible particle counts from MFI after 1 month of storage at 25 ℃. Sub-visible particle counts obtained from HIAC or after storage at other temperatures showed similar trends to the data set shown. Visual inspection did not indicate a high visible particle count for any of the samples, except for samples containing calcium chloride. High protein concentration solutions (. Gtoreq.20 g/L) are robust to withstand transport as long as some PS-20 is present. Thus, for solutions of 20-100g/L, trehalose formulations were used in long-term stability studies.

Shipping data confirmed that low concentration formulations were not robust as observed by high and highly variable sub-visibility particle counts. The data also show that the problem is not solved by polysorbate alone, so low concentration solutions need to be reconstituted.

d. Reconstitution of Low concentration DS

The low concentration reformulation screen was pressurized by simulated transport and passed the MFI test. The sub-visibility particle counts shown are >10 μm particles filtered by aspect ratio; the trend is similar for other particle sizes.

During the manufacturing process, high concentrations (. Gtoreq.100 g/L) of unformulated drug product (UDS) containing trehalose will be produced and frozen. Storage of this high concentration intermediate is necessary to enable dilution into different formulations spanning the 2-100mg/mL formulation range. Dilution by UDS may result in some trehalose remaining; for uniformity of the composition, a single trehalose concentration will be used for the entire low dose range. Buffer and pH were unchanged.

The primary screen was used to determine the minimum protein concentration at which the trehalose formulation was stable and the effect of increased ionic strength was determined by formulating in 150mM trehalose and 75mM arginine hydrochloride or calcium chloride. The results shown below indicate that protein concentrations of ≧ 10g/L are stable, but for robustness, the low concentration range was set to 2-20g/L. Increasing the ionic strength produced a more stable solution in both excipient cases. See, for example, fig. 8.

The next studies focused on optimizing arginine or NaCl concentrations; for all studies, a protein concentration of 2g/L was used as the worst case. For each excipient, the wide and narrow excipient concentration screens were run at 0.02% PS-20. The initial arginine screen was run with varying amounts of trehalose, where the solution was prepared by mixing 270mM arginine with 250mM trehalose. The aim was to maintain the osmolality, but the calculation was wrong (arginine hydrochloride is divalent), so the solution containing arginine was hypertonic. The remaining excipient concentration screens were performed at constant trehalose concentration at 40 or 50mM based on residual trehalose at 20g/L after dilution from 100g/L stock. The results are provided in fig. 9 and 10.

Broad arginine and NaCl screening results indicated that concentrations greater than 50mM arginine or 75mM NaCl were required in order to produce stable formulations. Narrow concentration screens of 75-150mM arginine or 100-200mM NaCl resulted in low particle counts throughout the range. This indicates that concentrations in the middle of these ranges should produce robust formulations; 130mM was selected to bind 50mM residual trehalose isotonic.

The same method was used to check PS-20 concentration optimization for new formulations. These experiments were performed simultaneously with narrow excipient concentration screens, so a midpoint concentration was used. Test conditions were 0.01-0.1% PS-20, 115mM arginine hydrochloride, 40mM trehalose; and 0.01-0.05% by weight of PS-20, 150mM NaCl,50mM trehalose. Several samples were tested with 0.02-80% PS, but yielded higher counts than the corresponding PS-20 results (data not shown). The particle count was low for all polysorbate levels tested, indicating that it was not necessary to change the level from 0.02% in the new formulation. See, for example, fig. 11.

The low concentration of anti-IL 5R reformulation results indicate that arginine or NaCl can both make the formulation stable in a short period of time. For protein concentrations of 2-20g/L, the formulation considered was 130mM arginine hydrochloride, or 130mM NaCl, with 50mM trehalose, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.

e. Vial and PFS considerations

The three formulations formed above (20-100 mg/mL in trehalose, 2-20mg/mL in trehalose/arginine and 2-20mg/mL in trehalose/NaCl) were appropriate for both vial and PFS configurations. The vial configuration was a 3cc Schott vial with a 4432/50West stopper. The greatest risk associated with vial placement is the level of silicone oil on the stopper, so long-term stability studies have silicone oil levels (0.039 mg/cm) ² ) Higher than the level of silicone oil commonly used (0.007-0.024 mg/cm) ² ) Is performed by the plug of (1).

The syringe tested for the anti-IL 5R formulation was a flat-bed syringe, i.e., BD 1mL long PFS with a chamfered flange, staked 29G thin-tube needle, containing 0.4mg silicone oil, and covered with a BD260 rigid needle shield (catalog No. 47363119).

f. Long term stability study

Two long-term stability studies were performed to validate the decisions made from the screening study. Stability study No. 1 investigated the long-term stability of PFS and trehalose and arginine/trehalose formulations in vials. Additionally, a PFS comparison is performed, which is not discussed here. Stability study No. 2 was initiated against the configuration examined in study No. 1 to provide data from another batch of material, and the effect of the NaCl/trehalose formulation interval and fill volumes from 1/2mL to 1mL in PFS and vials were also investigated.

i.1 stability study: anti-IL 5RPFS stability Explanation

Stability studies were performed in syringes using vials as controls. Each end point of the formulation interval was tested in each primary container. The syringe tested was the platform Hypak ^TM for Biotech; this is a 1mL long BD glass syringe, containing almost no tungsten, with a 29G thin-tube needle and 0.4mg silicone oil. The vial used was a 3cc Schott vial with a West 4423/50 stopper and seal.

The filled formulations were the following:

2 and 20mg/mL anti-IL 5R antibody, 125mM arginine hydrochloride, 50mM trehalose, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6; and

20 and 100mg/mL anti-IL 5R antibody, 250mM trehalose, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.

A. Purity specification for anti-IL 5R pre-filled syringes

There is no significant effect of the primary vessel on monomer loss for any formulation. Some effect of protein concentration was observed, but monomer loss rate was as low as ever. See fig. 12A.

B. Description of particle analysis of anti-IL 5R prefilled syringes

Particle formation is considered to be the major degradation pathway for anti-IL 5R, so it is considered to play a significant role in determining a suitable PFS. Sub-visible particle measurements by HIAC showed an increase in the number of particles in the PFS, which could be attributed to the silicone oil droplets (see, fig. 12B and 12C). However, the total particle count is still well below the USP limit, i.e. 6000 >10 μm particles/mL and 600 >25 μm particles/mL for all configurations. MFI was used as an orthogonal method and showed similar results, although less difference was observed between the vessels, as the silicone oil droplets could be filtered out of the results in the MFI software.

ii.at 5 ℃ under Hypak ^TM Overview of stability in for Biotech syringes

Table 1 shows the available Hypak at 5 deg.C ^TM Summary of stability data (stability study No. 1) for anti-IL 5R in a for Biotech syringe for up to 16 months (arginine formulation) and 24 months (trehalose formulation). Visible particles were detected, which resulted in a decrease in PS-20 concentration from 0.02% to 0.006%. No other high risk was identified, but the sub-visibility particle count was variable.

TABLE 1

Stability of anti-IL 5R in various formulations in Hypak for Biotech syringes at 5 ℃ for 16 months is summarized. The appearance results included particles, which were mitigated by reducing the PS-20 concentration (referred to in separate reports). Sub-visibility particle results were variable, but no trend was observed. All other results were within expectations of stable products. *

iii.2 stability study

Stability study No. 2 is the same stability study in pre-filled syringes and vials designed to determine low dose formulations and fill volumes, as well as to verify placebo stability.

The stability study previously used to select for PFS had only a 1mL fill volume. However, there are several potential benefits to lower fill volumes, including reduced injection pain, reduced subcutaneous bumps, more rapid dosing, and leakage from the site of administration.

The interval strategy for the 1mL fill option is two protein concentration intervals, 2-20g/L for low doses and 20-100g/L for high doses. The same interval for 1/2mL filling covers a dose range of 1-50mg, and also requires a 100g/L filling volume interval from 1/2mL to 1mL for a 50-100mg dose. This result is illustrated in fig. 13 for clarity. In both cases, the lowest dose interval was studied in formulations based on arginine and NaCl.

The filled drug and placebo were as follows:

all solutions: 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.0

2Arg/20Arg:2 or 20g/L anti-IL 5R,125mM arginine hydrochloride, 50mM trehalose

2NaCl/20NaCl:2 or 20g/L anti-IL 5R,130mM NaCl,50mM trehalose

20Tre/100Tre:20 or 100g/L anti-IL 5R,250mM trehalose

Arg placebo: 125mM arginine hydrochloride, 50mM trehalose

NaCl placebo: 130mM NaCl,50mM trehalose

Tre placebo: 250mM trehalose

The 1mL fill volume configuration was not placed in the test for any vial configuration or placebo in the PFS in order to reduce the overall size of the study, since the 1/2mL fill volume is most likely the worst case configuration due to the higher surface area-to-volume ratio.

All these samples were shipped and then placed in stability studies at 5 ℃,25 ℃ and 40 ℃. The vials were stored upside down during the stability study to minimize contact with the stoppers. When visible particles were found in previous studies, 6 weeks of data have been collected for this stability study. Additionally, particles were observed in the NaCl formulation between the month 3 and month 6 time points of this study (data not shown), thereby resulting in the NaCl formulation being excluded while the arginine formulation was supported.

Additional formulation work required to mitigate visible particle formation is described in the examples below and results in a reduction in polysorbate-20 concentration from 0.02% to 0.006%. No other stability issues were observed for this formulation, and no significant effect on container or fill volume was observed. One year stability data is summarized in fig. 14, including visible particle fraction, purity loss by SEC, particle count by HIAC, and potency (not all configurations were tested at all time points).

Conclusion of example 1

Formulation screening was performed using different stress methods including freeze/thaw, agitation, silicone oil incorporation, and accelerated stability. Results of the screening study were validated using long-term stability. The stage 2b trehalose formulation was successful for high concentration liquids with increased polysorbate concentrations, but showed instability when extended to low concentration liquids, primarily due to the formation of sub-visible particles. Reformulation of the low concentration range by increasing ionic strength with arginine hydrochloride or sodium chloride results in a stable solution.

To cover the wide range of possible doses (2-100 mg), three potential formulation intervals are as follows:

2-20g/L,130mM arginine hydrochloride, 50mM trehalose dihydrate, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.0;

2-20g/L,130mM sodium chloride, 50mM trehalose dihydrate, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.0; and

20-100g/L,250mM trehalose dihydrate, 20mM histidine/histidine hydrochloride, 0.02% PS-20, pH 6.0.

Long-term stability for up to 24 months indicates that the three formulations are stable at 2-8 ℃ with respect to agitation, relatively insensitive to silicone oils, and compatible with vials and PFS. In addition, minimal degradation was observed at elevated temperatures. Looking continuously at the two low concentration formulations, the NaCl option was eliminated due to increased visible particle formation compared to the arginine option, resulting in two formulation intervals. The data indicate that pre-filled syringes are acceptable primary containers for 1mL or 1/2mL fill volumes. Visible particle formation remains a problem for these formulations, which is solved as in the examples below.

Example 2 particle formation in anti-IL 5R formulations

In a previous long-term stability study, visible particles were detected in vials and aqueous anti-IL 5R formulations in PFS, with the first detection occurring at the 6 month time point (data not shown). Studies were conducted to mitigate visible particle formation in long-term storage anti-IL 5R antibody formulations comprising polysorbate 20 (PS-20). The following examples describe long-term studies of formulations containing different PS-20 and protein concentrations to verify the importance of PS-20 concentration in mitigating particle formation. This study resulted in an acceptable 0.002% -0.01% PS-20 range.

Abbreviations and Definitions

Preliminary study

Particle formation was detected in the long-term stability study described in example 1. However, the particles are extremely small and appear more like a cloud than individual particles. To see the particles, the sample was examined close to the light source. Since these particles are different from those in vials and PFS standards, these samples were compared to each other and to the standards at each time point.

Particles were initially detected at the 6 month time point in 100g/L Tre vials with 0.02% PS-20.

The 6 month long and 11 month long samples were compared in (1) vials and (2) pre-filled syringes (PFS). Both studies showed that particle formation in vials was more severe than in PFS, but PFS standards were not available at the time so multiple comparisons could not be made. Samples in the PFS were decanted and injected into vials for appearance testing. No more particles were observed in these vials, which verifies that the difference is not a path length effect. In the trehalose formulation, this vial-PFS difference was more pronounced than in the arginine formulation.

Different PS-20 concentrations (0, 0.01%, 0.02% and 0.03%) in 100g/L Tre formulation in vials and PFS after delivery were compared. At month 11, lower PS-20 concentrations of 0 and 0.01% PS-20 were completely particle free. Particles were visible in both the 0.02% and 0.03% PS-20 vials and PFS. At

month

20, 0 and 0.01% PS-20PFS and 0% PS-20 vials remained clear, but the 0.01% vials had very small amounts of rolled particles.

In the targeting study, the particles were clearly visible in the 100g/L Tre,0.02% PS-20 vials and PFS, when kept close to the light source, in some cases as early as 3-4 months. At the low concentration (20 g/L) end of the Tre interval, particle formation was much slower and was only observed in the vial at month 21. No particles were observed in any PS-20 concentration of 20g/L Tre PFS, and data was available for up to 21 months. Particle formation in the arginine formulation was somewhat slower, with particles observed at the high concentration (20 g/L) end of the interval at months 6-9 with 0.02% PS-20. No particles were observed in any PS-20 concentration of 2g/L Arg formulation in either vessel, and data was available for up to 16 months.

Acceleration and stress temperature studies do not provide any indication of particle formation. At 40 ℃, no particles were formed at all within a test period of 3 months. At 25 ℃, particle formation was similar or slightly reduced compared to 5 ℃.

This data indicates that the high protein concentration end points of the interval are at high risk for the presence of visible particles, while the low end points are potentially at risk in the long run. It was investigated that reducing the PS-20 concentration mitigates particle formation. The primary vessel is still the platform PFS, but vials are used as the early particle formation read data, since particle formation in vials is more severe and more readily visible.

Investigation of different PS-20 concentrations

An extended stability study was designed to investigate particle formation in different formulations, different PS-20 concentrations, and different antibody concentrations. All samples were filled into PFS and vials, transported twice to separate locations to simulate the dispensing process, and placed in a 5 ℃ stability study. The appearance test was performed once a month. Additional SEC, HIAC and MFI tests were performed on the sub-groups of samples at time zero, month 3, month 6 and month 9. Figure 15 shows a sample prepared as part of this study.

As shown in figure 16 for MFI data of PFS versus time zero, without any PS-20, sub-visible particles formed after transport. The lowest PS-20 test level (0.002%) or more was sufficient to protect against shipping stresses. In addition, it was verified that in the tank using the down-scaling model, the lowest PS-20 concentration (0.002%) was sufficient to protect against DS transport stress (data not shown).

The appearance results of the particles show that there was significant particle formation in the vials above 0.01% ps-20, whereas in the PFS of 0.02% ps-20 there were some particles in both formulation intervals and less particles were observed at the lowest concentration end point of the two intervals. See fig. 17. For 100g/L Tre, particles were first observed at

month

3 and 20g/L Arg at month 6. The figure shows the effect of protein concentration and PS-20 concentration on particle formation at the latest time point, i.e. month 9. It should be noted that particle formation observations are all made close to the light source.

The appearance data set an upper limit of 0.01% for the PS-20 concentration, while the sub-visibility particle count set a lower limit of 0.002% (actual limit may be lower depending on available data). The midpoint of this acceptable range (0.006%) was set as the new PS-20 concentration.

The overall stability of PFS was confirmed for the interval endpoint samples, including up to 9 months for 2g/L Arg and 20g/L Arg, and up to 18 months for 20g/L Tre and 100g/L Tre. The following assays were tested:

a.0.002, 0.006 and 0.01% PS-20: appearance, HIAC, MFI, and SEC.

B. Only 0.006% PS-20: instron tensile strength tester, bioassay, bioanalyzer, RP, and cIEF.

All data indicate that the new formulations are stable and low risk. A summary of the data is shown in table 2.

TABLE 2

The data from example 2 indicate that a reduction of PS-20 concentration to 0.006% ameliorated particle formation and produced an antibody formulation product that was stable for at least 9 months (arginine formulation) or 18 months (trehalose formulation), with no indication of signs of impending damage.

Example 3

Orthogonal method for particle detection

The primary method of particle detection and quantification is appearance testing by visual inspection as exemplified in example 2. Visual inspection is variable for a number of reasons. In general, visual inspection varies due to the inherent variability of human senses, resulting in different outcomes for different individuals. Due to the very small size of these particles, their visibility is highly dependent on the amount of light, and they are different from the standards for vials and PFS. These factors increase the variability of results between time points and between analysts.

The orthogonal method was investigated to verify the appearance results. The particles formed in the trehalose formulation were too small to be seen alone, so the sub-visible particle approach was investigated. Different batches from week 2, and

months

2, 5 and 9 were compared by DLS, FC, HIAC and MFI for worst case samples (100 g/L, tre,0.02% ps-20, vial).

DLS was performed at 100g/L, resulting in an underestimation of the unreliability of the main peak and all peak sizes as expected. The large peaks of the samples of 2 months, 5 months and 9 months long were detected and their size was 1.4-2.2 μm. Even though the reported size is not reliable, the presence of the peak is correlated with visible particles. However, neither the reported particle size nor the intensity of the particle peak correlate with visual appearance results.

HIAC results were similar for all samples; no particles were detected.

The MFI counts of particles >10 μm and >25 μm were similar for all samples. However, MFI counts and FC counts for particles >1 μm and >2 μm tended to visual appearance results and increased with sample age. The results for these samples are shown in fig. 18.

Further experiments indicate that particles >1 μm as determined by MFI provide a more reliable trend with visual appearance than larger particles or FC counts (data not shown).

Similar experiments were performed on 20g/L Arg samples, showing visible particles, but none of these orthogonal methods successfully detected these particles. The particles formed in the arginine formulation appeared larger than those formed in trehalose and were seen as individual particles, which may be why they were not detected by the sub-visible particle method.

MFI was used in example 2 to verify the effect of PS-20 concentration. A comparison of MFI and appearance score for the 9 month time point of example 2 is shown in fig. 19. Along with additional measurements (not shown), this comparison indicates that particles are visible when the MFI count exceeds approximately 100,000 particles (> 1 μm)/mL. The MFI results provide additional support for the following conclusions: i.e., particle formation is mitigated by 0.002% -0.01% PS-20, especially in PFS. The vial data was considered worst case and was also stable at the target concentration of 0.006% ps-20.

Example 4

Stability study

Additional stability studies were conducted to investigate all configurations of previous studies with new target PS-20 concentrations (0.006%) and to ensure the confidence that the addition of these formulations was stable. The intervals include a protein concentration interval and a fill volume interval, and are illustrated in fig. 20.

The increase in the fill volume interval increases the dose range covered by the trehalose formulation. These additional configurations reduce the risks associated with arginine formulations, which are more risky because particles form more slowly and cannot be detected by orthogonal methods. The samples included in this stability study were:

0, 2 and 20g/L,1mL fill, arginine formulation, 0.006% PS-20;

0 and 2g/L,0.3mL fill, arginine formulation, 0.006% PS-20;

0, 20, 50 and 100g/L,1mL fill, trehalose formulation, 0.006% PS-20; and

0 and 20g/L,0.3mL fill, trehalose formulation, 0.006% PS-20.

The samples were shipped twice to off-site locations to simulate the distribution process and then placed in 5 ℃,25 ℃, and 40 ℃ stability studies.

Nine months of data were collected for the arginine formulation, and twelve months of data had been collected for the trehalose formulation. These results are consistent with historical data and no particles were observed in these samples until this time point. In addition, no tendency for sub-visible particles has been observed over time, although some moderately high hyper-bias values appear. The range of results for all analyses is shown in table 3.

TABLE 3

/>

43 of the 44 measurements fall within this range, but a hyper-bias of 347 particles/mL is also measured.

The data supports that this formulation interval is suggested for the full dose range. The trehalose interval is less risky than the arginine interval and is suggested for doses as low as 6 mg.

Conclusion of examples 2 to 4

Previous particle observations in long-term stability studies have led to investigations into formulation variables that can be altered to improve stability. The key variables, namely protein concentration and polysorbate concentration, were tested and analyzed. Based on the data presented above, allowable polysorbate ranges from 0.002% to 0.01% and a target of 0.006% were determined as optimal formulations for anti-IL 5R antibody formulations. This observation is supported by two stability studies, primarily visual appearance tests, with data available at 18 months and 12 months.

It was also observed that by using a volume interval of 0.3-1.0mL at 20mg/mL, a trehalose formulation in the range of 6-20mg could be used instead of an arginine formulation. It was found that trehalose formulations were more predictable than arginine formulations due to the larger data set and better detection.

Example 5

Additional purification developments focused on reducing Host Cell Proteins (HCPs) were performed in order to determine the effect of HCPs on particle formation. anti-IL 5R was purified as outlined in FIG. 21.

Flow-through 2D gel analysis of the protein a column indicated that several protein species were present. See, for example, fig. 22. The major impurities included about 60% Fab fragments, about 5% Light Chain (LC) and fragments, and about 40% non-anti-IL 5R associated Host Cell Protein (HCP), as determined by reverse phase mass spectrometry (RP-MS). Non anti IL5R related HCPs including Glutathione S Transferase (GST), fructose two phosphate aldolase, and two Nu acetone enzyme.

The flow-through of the protein a column was further passed through a protein L column to further separate (1) Fab fragments from (2) non-anti-IL 5R HCP. It was found that some of the material in the non-anti-IL 5R HCP was responsible for particle formation and was not Fab fragments (data not shown).

To determine which of the major HCPs caused particle formation, GST was first investigated. The effect of GST on particle formation was investigated by the following steps: (1) Selectively removing GST to determine the effect on particle formation; (2) analyzing the particle pellet to determine the presence of GST in the pellet; and (3) adding GST to the anti-IL 5R formulation to determine the effect on particle formation.

Preliminary evidence for specific GST removal using an affinity matrix made of glutathione-crosslinked agarose conjugates suggests that GST removal results in reduced particle formation (data not shown). Analysis of the pellet formed by protein a flux indicated the presence of a high concentration of GST in the pellet (data not shown).

GST was added to purified anti-IL 5R formulations lacking particles to determine the effect of GST on the particle formulation. GST was obtained from commercial sources (Prospec). GST was added to a formulation containing 50mg/mL anti-IL 5R, 20mM histidine hydrochloride buffer, 9% (w/v) trehalose, 0.02% PS-20, pH 6.0, resulting in GST concentrations of 3.8. Mu.g/mg and 7.6. Mu.g/mg. The samples were incubated at 38-42 ℃. The particles were observed by placing the sample under a light box. The incubation time required to observe particle formation depends on the level of GST present. For both the 3.8. Mu.g/mg and 7.6. Mu.g/mg GST spiked samples, particles were observed after 24 hours at 38 ℃ -42 ℃ (data not shown).

These results indicate that the addition of GST to anti-IL 5R formulations results in particle formation in anti-IL 5R formulations.

Example 6

Investigation of GST activity in different anti-IL 5R formulations purified by different means. As a preliminary, GST activity assay (biological field) was used to determine GST concentration to form a standard curve. GST catalyzes the formation of the thiol group of Glutathione (GSH) to an electrophilic compound such as 1-chloro-2, 4-dinitrobenzene (CDNB) to form a GS-DNB conjugate, which is detected at 340 nM. Thus, the increase in absorbance at 340nM is proportional to GST activity. Using this assay, GST concentrations were determined for different anti-IL 5R formulations (samples A-J) purified using different procedures. A correlation between GST concentration and particle formation was noted. The results are provided in table 4.

TABLE 4

* Some particles, but not many LLOQ = lower limit of quantitation.

This evidence confirms that the presence of GST correlates with particle formation.

Example 7

Different purification columns were investigated to identify the most effective method of reducing the concentration of GST in anti-IL 5R formulations. See table 5. GST concentrations were determined as outlined in example 7.

TABLE 5

Description of the invention	GST concentration (μ g/ml)
		CM product	5.935
HA products	1.255
		MabSelectSure elution product	<LLOQ
CaptoAdhere products	<LLOQ
		CEX product	9.228
CEX product	21.081

* Two separate batches of anti-IL 5R antibody were assayed for GST concentration.

Table 5 shows that both the protein a column (MabSelect Sure) and the mixed mode chromatography column (CaptoAdhere) successfully reduced GST concentration below detectable levels during purification of anti-IL 5R antibodies.

Example 8

Investigate the presence of GST in other antibody preparations. The presence of particles in these antibody preparations was also investigated. The results are presented in table 6 together with the evaluations.

TABLE 6

All of the various embodiments or options described herein may be combined in any or all variations. While the present invention has been particularly shown and described with reference to certain embodiments thereof, it will be understood by those of ordinary skill in the art that they have been presented by way of example only, and not limitation, and various changes in form and details may be made therein without departing from the spirit and scope of the present invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents.

Sequence listing

<110> Aslikang (Sweden) Co., ltd

<120> Stable aqueous antibody formulation

<130> IL5R-400WO1

<140>

<141>

<150> 61/895,143

<151> 2013-10-24

<160> 10

<170> PatentIn 3.5 edition

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Thr Ser Glu Asp Ile Ile Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Thr Ser Glu Asp Ile Ile Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Ala Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Lys Val Thr Ile Thr Ser Asp Arg Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Gly Arg Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Ala Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Lys Val Thr Ile Thr Ser Asp Arg Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Gly Arg Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

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Ser Tyr Val Ile His

1 5

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Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Arg Phe Lys

1 5 10 15

Gly

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Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp Tyr

1 5 10

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Gly Thr Ser Glu Asp Ile Ile Asn Tyr Leu Asn

1 5 10

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His Thr Ser Arg Leu Gln Ser

1 5

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Gln Gln Gly Tyr Thr Leu Pro Tyr Thr

1 5

Claims

1. A stable aqueous antibody formulation that is not subject to freezing temperatures, comprising:

a. 30mg/mL of an antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 5-7, and wherein the light chain variable region comprises CDR1, CDR2, and CDR3 sequences defined by kabat of SEQ ID NOs 8-10; and

b. 0.006% polysorbate-20; and

c. 20mM histidine/histidine HCl; and

d. 250mM trehalose, wherein the formulation is pH 6.0.

2. The antibody formulation of claim 1, wherein the formulation is an injectable formulation, wherein the formulation is suitable for intravenous, subcutaneous, or intramuscular administration.

3. A sealed container containing the antibody formulation of claim 1 or 2, wherein the sealed container is a pre-filled syringe.

4. A pharmaceutical unit dosage form suitable for parenteral administration to a human comprising the antibody formulation of any one of claims 1-3 in a suitable container, wherein the suitable container is a pre-filled syringe.